Modified factor h binding proteins (fhbps) and methods of use thereof

Abstract

The present application relates to modified factor H binding proteins (FHBP) and methods of use thereof. Provided are factor H binding proteins and methods of use thereof that are capable of eliciting antibodies that are bactericidal against at least one strain of Neisseria meningitidis.

Description

[0001] This application is a divisional application of a divisional application filed on March 29, 2011, with Chinese application number 201510771883.0 and invention title "Modified Factor H Binding Protein (FHBP) and Method of Using the Same Thereof" (original application number 201180017429.6).

[0002] Cross-references

[0003] This application claims priority to U.S. Provisional Patent Applications No. 61 / 319,181, filed March 30, 2010; No. 61 / 334,542, filed May 13, 2010; No. 61 / 381,025, filed September 8, 2010; No. 61 / 423,757, filed December 16, 2010; and No. 61 / 440,227, filed February 7, 2011, the entire contents of which are incorporated herein by reference.

[0004] Statement on Federally Funded Research

[0005] This invention was completed with funding from the National Institute of Allergy and Infectious Diseases and the National Institutes of Health, under grant numbers R01 AI046464, R01 AI 082263, and AI 070955. The government holds certain rights to this invention.

[0006] Foreword

[0007] Neisseria meningitidis is a Gram-negative bacterium that resides in the human upper respiratory tract. Most notably, it causes sporadic and periodic outbreaks of meningitis and sepsis worldwide. Morbidity and mortality are highest in children under two years of age. Similar to other Gram-negative bacteria, Neisseria meningitidis typically possesses a cytoplasmic membrane, a peptidoglycan layer, an outer membrane, and pili. The outer membrane, along with capsular polysaccharides, forms the bacterial cell wall, and the pili protrude into the external environment. Encapsulated Neisseria meningitidis strains are a leading cause of bacterial meningitis and sepsis in adolescents. The prevalence and economic importance of invasive Neisseria meningitidis infections have spurred research into effective vaccines that provide immunity against various strains, particularly the genetically diverse serotypes or subtypes of serogroup B strains.

[0008] Factor H binding protein (fHbp, also known in the art as lipoprotein 2086 (Fletcher et al. (2004) Infect Immun72:2088-2100), genome-derived Neisseria genus antigen (GNA) 1870 (Masignani et al. (2003) J Exp Med197:789-99) or "741") is a meningococcal protein expressed in bacteria as a surface-exposed lipoprotein. A key function of fHbp is its binding to human complement factor H (fH), which downregulates complement activity. The binding of fH to the bacterial surface is a crucial mechanism by which pathogens survive in unimmunized human serum or blood and evade innate host defenses. Recently, genetic variations in the human factor H gene cluster have been found to influence susceptibility to meningococcal disease (Davila S et al. (2010) Nat Genetics doi:10.1038 / ng.640). The specific binding of fH to human fH fHbp could explain why Neisseria meningitidis is strictly considered a human pathogen.

[0009] There is still a need for an fHbp peptide that can elicit an effective bactericidal antibody response. Invention Overview

[0010] This application provides an H factor binding protein and a method of using the protein, which can induce bactericidal antibodies against at least one strain of Neisseria meningitidis.

[0011] This application relates to the following.

[0012] 1. A non-naturally occurring H factor binding protein (fHbp), said protein having a lower affinity for human H factor (fH) than fHbp ID 1, wherein said non-naturally occurring fHbp has a conformational epitope for antibody binding, said antibody having bactericidal activity against at least one strain of Neisseria meningitidis.

[0013] 2. The non-naturally occurring fHbp according to claim 1, wherein the fHbp contains at least one amino acid substitution compared to the naturally occurring control fHbp.

[0014] 3. The non-naturally occurring fHbp as described in claim 1, wherein the affinity of the fHbp for binding to human fH is 50% or less of the affinity of fHbpID1 for human fH.

[0015] 4. The non-naturally occurring fHbp according to claim 1, wherein the affinity of the fHbp for binding to human fH is less than 40%, 30%, 20%, 10%, 5%, 2%, 1%, 0.1%, or 0.1% of the affinity of fHbpID1 for human fH.

[0016] 5. The non-naturally occurring fHbp as described in claim 1, wherein the serum level of the bactericidal antibody induced by said fHbp is at least as high as the level induced by fHbp ID 1.

[0017] 6. The non-naturally occurring fHbp as described in claim 5, wherein the serum level of the bactericidal antibody induced by said fHbp is at least twice as high as the level induced by fHbp ID 1.

[0018] 7. The non-naturally existing fHbp according to item 1, wherein the fHbp is derived from variant 1fHbp.

[0019] 8. The non-naturally existing fHbp according to item 1, wherein the fHbp is derived from the variant 2fHbp.

[0020] 9. The non-naturally occurring fHbp according to claim 1, wherein the fHbp is derived from the variant 3fHbp.

[0021] 10. The non-naturally occurring fHbp according to claim 1, wherein the fHbp comprises a variable segment V A V C , and V E All of them are derived from lineage 1.

[0022] 11. The non-naturally existing fHbp as described in item 7, wherein the fHbp is derived from module group I fHbp or module group IV fHbp.

[0023] 12. The non-naturally existing fHbp according to item 8, wherein the fHbp is derived from module group III fHbp or module group VI fHbp.

[0024] 13. The non-naturally existing fHbp according to item 9, wherein the fHbp is derived from module group II fHbp or module group V fHbp.

[0025] 14. The non-naturally existing fHbp according to item 1, wherein the fHbp is derived from module group VII, VIIII, IX, or X fHbp.

[0026] 15. The non-naturally occurring fHbp according to claim 1, wherein the fHbp is an artificially prepared chimera.

[0027] 16. The non-naturally occurring fHbp according to claim 15, wherein the fHbp is derived from the amino acid sequence of chimeric I.

[0028] 17. The non-naturally occurring fHbp according to claim 1, wherein the fHbp contains an amino acid substitution at a position corresponding to amino acid residues 41, 60, 80, 113, 114, 117, 119, 121, 128, 130, 147, 148, 149, 178, 195, 199, 211, 220, 222, 236, 241, 247, or 248, the numbering being based on the ID 1 number of mature fHbp.

[0029] 18. The non-naturally occurring fHbp according to claim 1, wherein the fHbp contains an amino acid substitution at a position corresponding to amino acid residues 87, 109, 115, 118, 126, 138, 197, 201, 202, 203, 209, 217, 225, 235, or 245, the numbering being based on the ID 1 number of mature fHbp.

[0030] 19. The non-naturally occurring fHbp according to item 17, wherein the fHbp contains an amino acid substitution at the position corresponding to amino acid residue 41.

[0031] 20. The non-naturally occurring fHbp according to claim 19, wherein the fHbp contains R41S as a substitution for the amino acid.

[0032] 21. The non-naturally occurring fHbp according to claim 19, wherein the fHbp contains R41A as a substitution for the amino acid.

[0033] 22. The non-naturally occurring fHbp according to claim 17, wherein the fHbp contains R130A as a substitution for the amino acid.

[0034] 23. The non-naturally occurring fHbp according to claim 17, wherein the fHbp contains H119A as a substitution for the amino acid.

[0035] 24. The non-naturally occurring fHbp according to claim 17, wherein the fHbp contains E218A or E239A as a substitution for the amino acid.

[0036] 25. The non-naturally existing fHbp according to any one of items 19-24, wherein the fHbp is derived from fHbp ID1.

[0037] 26. The non-naturally occurring fHbp according to claim 17, wherein the fHbp comprises fHbp ID 14, and the fHbp ID 14 contains R41S as a substitution for the amino acid.

[0038] 27. The non-naturally occurring fHbp according to claim 17, wherein the fHbp comprises fHbp ID 4, and the fHbp ID 4 contains R41S as a substitution for the amino acid.

[0039] 28. The non-naturally occurring fHbp according to claim 17, wherein the fHbp comprises fHbp ID 9, and the fHbp ID 9 contains R41S as a substitution for the amino acid.

[0040] 29. The non-naturally occurring fHbp according to claim 17, wherein the fHbp comprises fHbp ID 74, and the fHbp ID 74 contains R41S as a substitution for the amino acid.

[0041] 30. The non-naturally occurring fHbp according to claim 17, wherein the fHbp is fHbp ID 15, and the fHbp ID 15 contains S41P as a substitution for the amino acid.

[0042] 31. The non-naturally occurring fHbp according to claim 17, wherein the fHbp contains R80A as a substitution for the amino acid.

[0043] 32. The non-naturally occurring fHbp according to claim 17, wherein the fHbp contains D211A as a substitution for the amino acid.

[0044] 33. The non-naturally occurring fHbp according to claim 17, wherein the fHbp contains E218A as a substitution for the amino acid.

[0045] 34. The non-naturally occurring fHbp according to claim 17, wherein the fHbp contains E248A as a substitution for the amino acid.

[0046] 35. The non-naturally occurring fHbp according to claim 17, wherein the fHbp contains G236I as a substitution for the amino acid.

[0047] 36. The non-naturally occurring fHbp according to claim 17, wherein the fHbp contains an amino acid substitution at one or both positions of 220 and 222.

[0048] 37. The non-naturally occurring fHbp according to claim 17, wherein the fHbp comprises T220A and H222A as said amino acid substitutions.

[0049] 38. The non-naturally existing fHbp according to any one of items 31-37, wherein the fHbp is derived from fHbp ID22.

[0050] 39. The non-naturally occurring fHbp according to claim 17, wherein the fHbp contains an amino acid substitution at one or more of positions 41, 113, 119, and 121.

[0051] 40. The non-naturally occurring fHbp according to claim 17, wherein the fHbp contains R41S and K113A as substitutions for the amino acid.

[0052] 41. The non-naturally occurring fHbp according to claim 17, wherein the fHbp contains R41S and K119A as substitutions for the amino acid.

[0053] 42. The non-naturally occurring fHbp according to claim 12, wherein the fHbp contains R41S and D121A as substitutions for the amino acid.

[0054] 43. The non-naturally occurring fHbp according to claim 12, wherein the fHbp comprises R41S, K113A, and D121A as the amino acid substitutes.

[0055] 44. The non-naturally existing fHbp according to any one of items 39-43, wherein the fHbp is derived from fHbp ID77.

[0056] 45. The non-naturally occurring fHbp according to claim 17, wherein the fHbp is derived from the amino acid sequence of chimeric I and contains R41S as a substitution for the amino acid.

[0057] 46. ​​The non-naturally occurring fHbp according to claim 17, wherein the fHbp is fHbp ID 28, and the fHbp ID 28 contains E218A and / or K199A as the amino acid substitution.

[0058] 47 An immunogenic composition comprising:

[0059] a) fHbp as described in any one of items 1-46; and

[0060] b) Pharmaceutically acceptable excipients.

[0061] 48. An immunogenic composition comprising:

[0062] a) An isolated H factor binding protein (fHbp) comprising at least 85% amino acid sequence identity with fHbp ID 4, fHbp ID 14, and fHbp ID 15, wherein the fHbp has a lower affinity for human H factor (fH) than fHbp ID 1; and

[0063] b) Pharmaceutically acceptable excipients.

[0064] 49. The immunogenic composition according to item 47 or 48, wherein the fHbp is in a vesicle formulation prepared from a strain of Neisseria meningitidis.

[0065] 50. The immunogenic composition according to item 47 or 48, wherein the pharmaceutically acceptable excipient comprises an adjuvant.

[0066] 51. The immunogenic composition according to item 47 or 48 further comprises NspA.

[0067] 52. A method for inducing an antibody response in a mammal, the method comprising administering to the mammal fHbp according to any one of claims 1-46, or an immunogenic composition according to claim 47 or 48.

[0068] 53. The method according to item 52, wherein the mammal is a human.

[0069] 54. The method according to claim 52, wherein the administration is for generating bactericidal antibodies against Neisseria meningitidis.

[0070] 55. A nucleic acid, said nucleic acid encoding fHbp according to any one of items 1-46.

[0071] 56. A recombinant expression vector comprising the nucleic acid described in claim 55.

[0072] 57. A genetically modified host cell comprising the nucleic acid of claim 55 or the recombinant expression vector of claim 56.

[0073] 58. An immunogenic composition comprising:

[0074] a) Vesicles derived from genetically modified Neisseria host cells, said cells being genetically modified with a nucleic acid encoding fHbp according to any one of claims 1-46, such that the genetically modified host cells produce the encoded non-naturally occurring fHbp, wherein said vesicles contain the encoded non-naturally occurring fHbp; and

[0075] b) Pharmaceutically acceptable excipients.

[0076] 59. The immunogenic composition according to claim 58, wherein the vesicles are natural outer membrane vesicles.

[0077] 60. The immunogenic composition according to claim 58, wherein the affinity of fHbp for binding to human fH is 40%, 30%, 20%, 10%, 5%, 2%, or 1% of the affinity of fHbpID1 for human fH.

[0078] 61. The immunogenic composition according to claim 58, wherein the host cell is genetically modified to provide a polypeptide product with reduced or inactive activity of the lpxL1 gene and / or lpxL2 gene.

[0079] 62. The immunogenic composition according to claim 58, wherein the host cells are genetically modified to increase NspA expression.

[0080] 63. A method for inducing an antibody response in a mammal, the method comprising administering to the mammal the immunogenic composition according to claim 58.

[0081] 64. A method for determining the likelihood of an H factor binding protein (fHbp) eliciting a bactericidal response against at least one strain of Neisseria meningitidis in an individual, the method comprising:

[0082] The ability of an antibody to inhibit the binding of human factor H (fH) to fHbp, said antibody being present in serum obtained from an individual immunized with fHbp, wherein the level of inhibition of fH binding to fHbp by said antibody is at least about 25% higher than the level of inhibition of fH binding to fHbp by a control antibody that inhibits the binding of fH to fHbp but does not produce a bactericidal response, indicates that fHbp may elicit a bactericidal response against at least one strain of Neisseria meningitidis.

[0083] 65. The method according to item 64, wherein the fHbp is a non-naturally occurring fHbp that has an affinity for fH lower than fHbp ID 1.

[0084] 66. A method for determining the likelihood that a non-naturally occurring factor H binding protein (fHbp) induces bactericidal antibodies against at least one strain of Neisseria meningitidis in an individual, said fHbp having an affinity for human factor H (fH) lower than fHbp ID 1, said method comprising:

[0085] To determine the ability of antibodies induced by non-naturally occurring fHbp to inhibit the binding of fH to fHbp in non-human test animals.

[0086] The antibody induced by the non-naturally occurring fHbp showed an inhibitory level of at least about 25% higher inhibition of fH binding to fH than the antibody induced by fHbp ID 1 in non-human test animals, indicating that the non-naturally occurring fHbp has the potential to elicit a bactericidal response against at least one strain of Neisseria meningitidis. Brief description of the attached diagram

[0087] Figure 1Figure A shows the standard curve of human fH concentration obtained by ELISA using meningococcal fHbp as the antigen in each well. See Example 1 for details. Figure B shows the human fH concentration in the serum of human fH transgenic (Tg) mice, including human fH-Tg negative littermates or known wild-type BALB / c mice, as well as the human fH concentration in human serum. See Example 1.

[0088] Figure 2 Following immunization with a group C meningococcal conjugate control vaccine, serum IgG antibody responses (insets A and B) in human fH transgenic (fH Tg) BALB / c mice and wild-type (WT) BALB / c mice, and serum bactericidal titers against group C 4243 strain (inset C). The conjugate vaccine does not bind to human fH. See Example 1 for details. Inset D: Human fH binds to the wild-type fHbp vaccine, but not to the control MenC-CRM conjugate vaccine or certain mutant fHbp vaccines, as illustrated in the corresponding figures in Table 5 of the Examples section. Mouse (or rabbit, or rat, etc.) fH does not bind to wild-type fHbp.

[0089] Figure 3 The relationship between serum human fH concentration and serum bactericidal antibody response in fH transgenic mice after inoculation with wild-type fHbp bound to human fH (inset A) or R41S mutant not bound to human fH (inset B). Inset C shows the GMT ratio (correlation between mutant / wild-type vaccine and serum human fH concentration in immunized fH transgenic mice) estimated using a general linear regression model. See Example 4 for details.

[0090] Figure 4 The binding of human fH, anti-fHbp mAb, JAR4, and JAR5 to wild-type and mutant fHbp (a mutant of fHbpID1 containing Glu replaced by Ala) was determined using enzyme-linked immunosorbent assay (ELISA).

[0091] Figure 5 SDS-PAGE size and purity analysis of WT fHbp ID 1 and ID 1 double mutants E218A / E239A. Molecular weights in kDa are shown on the left.

[0092] Figure 6 The inhibitory effect of soluble fHbp on the binding of antagonistic fHbp MAb to immobilized wild-type fHbp was detected by ELISA.

[0093] Figures 7A-7D : Figure 7A and 7B Showing wild-type fHbp ID 1 and E218A / E239A double mutant protein ( Figure 7A ) and wild-type fHbp ID 1 and R41S mutant protein ( Figure 7B The results of differential scanning calorimetry. Figure 7C The titers of anti-fHbp IgG antibodies in mice immunized with wild-type fHbp ID 1 or E218A / E239A double mutant protein are shown by ELISA. Anti-fHbp IgG antibody responses in mice immunized with WT or mutant fHbp are also shown. In Study 3, mice were immunized with three doses of recombinant WT or mutant fHbp adsorbed on Freund's adjuvant (FA) or aluminum hydroxide (Al(OH)3); in Study 4, CD-1 mice were immunized with one dose of WT or mutant fHbp adsorbed on aluminum hydroxide (Al(OH)3); and in Study 5, BALB / c mice were immunized with three doses of WT or mutant fHbp adsorbed on aluminum hydroxide (Al(OH)3). Shaded bars for WT fHbp; hollow bars for E218A / E239A mutant fHbp. Figure 7D The titer of anti-fHbp IgG in BALB / c mice from Study 6, which were given two doses of the fHbp vaccine, is indicated.

[0094] Figure 8 Figure A shows the binding of fH to the natural fHbp variant. Each well of the ELISA plate is coated with recombinant fHbp representing variant fHbp ID 1, 14, or 15. Binding of human fH is detected as described in the Examples section. Figures B and C show the binding of variants fHbp ID 1, 14, and 15 to Mab JAR4 and JAR5, respectively.

[0095] Figures 9A-9B : Figure 9A The structure of the fHbp complex with the human fH fragment is shown in the cartoon illustration. In the cartoon, fHbp is represented in black at the bottom, and fH is represented in gray at the top. The structural model of the fHbp-fH fragment combination is based on published atomic coordinates (Schneideret et al. ((2009) Nature 458:890-3)). The black bands represent the N- and C-terminal domains of the fHbp molecule, respectively. The gray bands represent the previously published sixth and seventh short homologous repeat domains of human fH, which mediate the interaction between human fH and fHbp (Schneideret et al. ((2009) Nature 458:890-3). The enlarged view on the left shows the arginine residue at position 41, which forms a charged H bond with fH. It is expected that the H bond will disappear after arginine is replaced by serine (bottom right inset). This figure was generated using MacPyMol (www.pymol.org). Figure 9BThe amino acid sequence of human H factor (fH) is shown. Its GenBank accession number is NP_000177 (P08603), and its encoding nucleic acid is NM_000186.

[0096] Figure 10 The binding of fH (small figure A) or anti-fHbp Mab JAR 4 (small figure B) or JAR 5 (small figure C) to the R41S and R41A mutants of fHbp ID 1 was detected by ELISA. The binding detection of human fH (small figure A) to anti-fHbp MAb (small figures B and C) is described in Example 2.

[0097] Figure 11 The binding of human H factor (left column) or anti-fHbp Mab JAR 4 (right column) to different fHbp variants and their corresponding R41S mutants in variant group 1. Binding detection is shown in Example 2. Insets A and B show the binding results for fHbp ID 4. Insets C and D show the binding results for fHbp ID 9. Insets E and F show the binding results for fHbp ID 74. "ID" refers to the fHbp amino acid sequence variant identification (ID) number, such as the Neisseria multilocus sequence classification website. http: / / pubmlst(dot)org / neisseria / fHbp / As described in [the text].

[0098] Figure 12 The binding of human fH and anti-fHbp MAb controls to the corresponding R41S mutants of fHbp and fHbp in variant group 2. Insets A and B show the binding of human H factor to the R41S mutants of wild-type (WT) fHbp ID 19 and fHbp ID 19. Insets C and D show the binding of human H factor to the R41S mutants of WT fHbp ID 22 and fHbp ID 22. Insets E and F show the binding of human H factor to the R41S mutants of WT fHbp ID 77 and fHbp ID 77. MAb controls are JAR 4 (insets B and D) or JAR 11 (inset F).

[0099] Figure 13The effect of anti-fHbp antibodies induced in the serum of human fH transgenic mice on the binding of fH to fHbp. The binding of fH to fHbp was detected by ELISA using 1:100 dilutions of pre-immunization (Figure A, pre-immunization) and post-immunization (Figure B, post-immunization) sera from transgenic mice immunized with either wild-type fHbp ID1 or R41S mutant ID1 fHbp vaccines. For the aluminum control group, hollow squares represent data from mixed sera from transgenic mice containing human fH, and closed triangles represent data from sera from wild-type mice without human fH. OD values ​​represent the amount of human fH bound, detected using sheep anti-human fH and donkey anti-sheep IgG conjugated to alkaline phosphatase. Figure C shows the anti-fHbp IgG titer in post-immunization serum, indicating similar antibody responses against both vaccines. Figure D shows the inhibition of human fH binding to fHbp in the presence of added human fH. Figure E shows the relationship between the percentage of fH binding inhibition and the SBA titer in human fH transgenic mice immunized with the fHbp vaccine.

[0100] Figure 14 Insets A and B show the binding of the K241E mutant of fHbp ID 1 to fH and to MAb JAR5, respectively. Insets C and D show the binding of the E241K mutant of fHbp ID 15 to fH and to MAb JAR5, respectively. Binding of fH or anti-fHbp MAb to fHbp was detected as described in Example 2.

[0101] Figure 15 The binding of fH or anti-fHbp MAb to the H119A and R130A single mutants of fHbp ID 1 was detected using ELISA. The binding of human fH (small figure A) to anti-fHbp MAb JAR 5 (small figure B) or JAR 4 (small figure C) was detected as described in Example 2.

[0102] Figure 16 Schematic diagram of the six most common fHbp module groups, named I through VI. Variable fragments are derived from one of two genetic lineages, named α (in gray) or β (in white). Following the nomenclature used at pubmlst.org / neisseria / fHbp / , the α and β lineages can also be named lineages 1 and 2. Fragment V A It begins at amino acid residue 8 and extends to position 73, while fragment V... B Starting at position 79 and extending to position 93 (the amino acid residues are numbered based on the sequence of fHbp ID 1). Fragment V C It begins at amino acid residue 98 and extends to position 159, while fragment V...D It starts at position 161 and extends to position 180. (Segment V) E Starting at amino acid residue 186 and extending to position 253. Of the 70 fHbp amino acid sequence variants analyzed, 33 contained only α-type fragments, and 7 contained only β-type fragments, designated as module groups I and II, respectively. The remaining 30 fHbp variants were natural chimeras containing different combinations of α and β fragments, which could be assigned to one of the four module groups (III-VI). The relationship between module groups and Masignani variant group nomenclature is shown, as well as the number of unique sequences observed in each fHbp module group. Figure 16 The last module diagram shows the module architecture of engineered (non-naturally occurring) fHbp chimeric I. For the engineered chimeric protein from fHbp ID 1 and ID 77 "chimeric I" (Beernink et al. (2008) Infec. Immun. 76:2568-2575), the four amino acid residues GEHT (SEQ ID NO:27) at positions 136 to 139 represent V C The nodes of the fragment (see Figure 19 ID refers to a publicly available website. http: / / pubmlst.org / neisseria / fHbp / The fHbp sequence peptide identification number described in the document.

[0103] Figure 17 The binding of fH to the recombinant fHbp mutant S41P (a mutant of fHbp ID 15). Inset A shows the binding of fH to the S41P mutant of fHbp ID 15. Insets B and C show the binding of the S41P mutant of fHbp ID 15 to MAbJAR 5 and MAbJAR31, respectively. "Pep28" represents fHbp ID 28; "Pep1" represents fHbp ID 1; "Pep 15WT" represents fHbp ID 15; and "Pep 15S41P" represents the S41P mutant of fHbp ID 15.

[0104] Figure 18 The binding of human fH to the R41S mutant of fHbp chimera I (Beernink et al. (2008) Infec. Immun. 76:2568-2575) (inset A) and the binding of the corresponding JAR 5 (inset B).

[0105] Figure 19Figure A shows the fHbp sequence alignment of the natural variant and the artificial chimera (chimera I; Beernink et al. (2008) Infec. Immun. 76:2568-2575). fHbp ID 1 belongs to module group I (according to the definition in Beernink and Granoff (2009) Microbiology 155:2873-83, all five variable segments, AE, are derived from the α lineage). fHbp ID 28 belongs to module group II (all five segments are derived from the β lineage). fHbp ID 15 is a natural chimera (belonging to module group IV, with one βA segment and αB, C, D, and E segments). The β-type A segment of fHbp ID 28 (V A Residues 8-73) and the corresponding A fragment (V) of fHbp ID 15 A In comparison, the latter also possesses a β-type A segment (V A β). Rectangles represent altered residues in the E218A / E239A double mutant fHbp. Inset B shows the alignment of fragment A (amino acid residues 8 to 73) for fHbp ID 1 and fHbp ID 77. Inset C shows the alignment of fragment C (amino acid residues 98 to 159) for fHbp ID 1 and fHbp ID 77. The node is at residue 136. Chimeric fHbp comprises the amino acid sequence from ID1 to residue G136, and the sequence of fHbp ID 77 from residue 136 to the C-terminus. Inset D shows the alignment of the native polymorphism at amino acid position 41 (numbered based on the numbering of fHbp ID 1); some variants have arginine (R41, ID 1, 19, 4, 9, and 74), while other variants have serine (S41, ID 55, 15) or proline (P41, ID 28). ID refers to the fHbp sequence ID; MG refers to the fHbp module group; and VG refers to the variant group. Figure E shows the alignment of fHbp ID 1, fHbp ID 77, and chimera I. The shaded residues in fHbp ID 77 highlight fragment V. C The residues in the middle are different from those in chimeric I. The residues in bold and shaded order from N-terminus to C-terminus correspond to K113, K119, and D121.

[0106] Figure 20 The binding of fH or anti-fHbp MAb to the K113A, K119A, and D121A single mutants of fHbp ID 77 was detected using ELISA. The binding of human fH (small figure A) and anti-fHbp MAb JAR 31 (small figure B) to the fHbp ID77 mutant was detected as described in Example 2.

[0107] Figure 21 The binding of fH or anti-fHbp MAb to the R41S / K113A, R41S / K119A, and 41S / D121A double mutants of fHbp ID 77 was detected using ELISA. The binding of human fH (inset A) and anti-fHbp MAb JAR 31 (inset B) to the fHbp ID 77 mutant was detected as described in Example 2.

[0108] Figure 22 The binding of fH or anti-fHbp MAb to the K113A / D121A double mutant and R41S / K113A / D121A triple mutant of fHbp ID 77 was detected using ELISA. The binding of human fH (small figure A), anti-fHbp MAb JAR 4 (small figure B), and anti-fHbp MAb JAR 31 (small figure C) to the fHbp ID 77 mutant was detected as described in Example 2.

[0109] Figure 23 The binding of fH to the fHbp ID 22 mutant was detected using ELISA. As described in Example 2, the binding of human fH to D221A, R80A, or wild-type fHbp was detected (Figure A); the binding of human fH to E218A, E248A, or wild-type fHbp was detected (Figure B); and the binding of human fH to R41S, Q38A, Q126A, or wild-type fHbp was detected (Figure C).

[0110] Figure 24 The binding of anti-fHbp MAb JAR31 to the fHbp ID 22 mutant was detected using ELISA. The binding of R80A and D211A mutants to JAR31 was detected as described in Example 2 (inset A), and the binding of E218A and E248A mutants to JAR31 was detected (inset B).

[0111] Figure 25 The binding of anti-fHbp Mab JAR4 to the fHbp ID 22 mutant was detected using ELISA. The binding of R80A and D211A mutants to JAR4 was detected as described in Example 2 (inset A), and the binding of E218A and E248A mutants to JAR4 was detected (inset B).

[0112] Figure 26 The binding of anti-fHbp MAb JAR35 to the fHbp ID 22 mutant was detected using ELISA. The binding of R80A and D211A mutants to JAR35 was detected as described in Example 2 (inset A), and the binding of E218A and E248A mutants to JAR35 was detected (inset B).

[0113] Figure 27 The binding of anti-fHbp MAb to the T220A / H22A double mutant or the G236I mutant of fHbp ID 22 was detected using ELISA. The binding of human fH (small figure A), anti-fHbp MAb JAR 31 (small figure B), JAR 35 (small figure C), or JAR 4 (small figure D) to the fHbp ID 22 mutant was detected as described in Example 2.

[0114] Figure 28 The binding of fH or anti-fHbp MAb to the R41S, Q38A, and A235G mutants of fHbp ID 22 was detected using ELISA. The binding of human fH (small figure A), anti-fHbp MAb JAR 31 (small figure B), or JAR 35 (small figure C) to the fHbp ID 22 mutant was detected as described in Example 2.

[0115] Figure 29 The binding of fH or anti-fHbp MAb to the Q126A, D201A, and E202A mutants of fHbp ID 22 was detected using ELISA. The binding of human fH (small figure A), anti-fHbp MAb JAR 35 (small figure B), and the fHbp ID 22 mutant was detected as described in Example 2.

[0116] Figure 30 : Binding with the fHbp ID 28 (variant group 3) mutant. Insets A and C show the binding of fH to the K199A, E217A, and E218A mutants detected by ELISA. Insets B and D show the binding of anti-fHbp MAb JAR 31 (inset B) and anti-fHbp MAb JAR 33 (inset D, only fHbp wild-type ID 28WT and E218A mutants) to the fHbp ID 28 mutant.

[0117] Figure 31 Serum bactericidal titers in wild-type BLAB / c mice immunized with an indicator mutant of the fHbp ID 1 vaccine were described, and titers were detected against serogroup B strain H44 / 76 (fHbp ID 1).

[0118] Figure 32Serum bactericidal titers in mice immunized with an indicator mutant of fHbp ID 22 were described, and titers were measured against serogroup W-135 strain Ghana 7 / 04 (fHbp ID 23). The top figure shows the mutant vaccine, which induced no significant difference in titer compared to the wild-type (WT) fHbp ID 22 vaccine (P>0.10). The bottom figure shows the mutant vaccine, which induced a significant decrease in titer compared to the control WT ID 22 vaccine (P<0.05).

[0119] Figure 33 The bactericidal titer in mice immunized with the R41S / K113A / D121A triple mutant of fHbp ID 77 was described, and the titer was measured against serogroup W-135 strain Ghana 7 / 04 (fHbp ID 23).

[0120] Figure 34 The amino acid sequences of fHbp ID 1 (SEQ ID NO:1), fHbp ID 22 (SEQ ID NO:2), fHbp ID 77 (SEQ ID NO:4), fHbp ID 28 (SEQ ID NO:3), and the ID1 / ID77 chimera (SEQ ID NO:8) were compared. ID 28 served as the control sequence for fHbp variant group 3. As described by Schneider et al. ((2009) Nature 458:890-3), based on the crystal structure of fHbp ID 1 complexed with the fH fragment, residues (highlighted in gray) that form hydrogen bonds or interionic interactions at the H factor binding interface were predicted. GEHT (SEQ ID NO:27) (bold) at positions 136 to 139 represents the junction between the chimera fHbp ID 1 and ID 77.

[0121] Figure 35 The amino acid sequences of fHbp ID 1 (SEQ ID NO:1), fHbp ID 22 (SEQ ID NO:2), fHbp ID 77 (SEQ ID NO:4), fHbp ID 28 (SEQ ID NO:3), and the ID1 / ID77 chimera (SEQ ID NO:8) were compared. Residues marked in gray indicate mutated residues, which are summarized in Table 7.

[0122] Figure 36 A model of fHbp in the complex formed with the fH fragment is described. The positions of amino acid residues known to affect the anti-fHbp mAbJAR 3 and JAR 5 (G121 and K122) and mAb 502 (R204) epitopes are described.

[0123] Figures 37A-37D Describes the use of ELISA ( Figure 37A Plasma resonance ( Figure 37B Flow cytometry targeting live bacteria Figure 37C only mAb; and Figure 37D The binding of human IgGa mouse chimeric fHbp-specific mAb to fHbp was detected in 20% of human serum with IgG removed.

[0124] Figures 38A-38B The study describes how human IgG1 mouse chimeric anti-fHbp mAb JAR 3, JAR 5, and mAb 502 activated complement and induced C1q-dependent C4b deposition in enveloped serogroup B bacteria H44 / 76. Figure 38A Serum from individuals with C1q depletion was used as a complement source; Figure 38B Before the reaction, purified C1q protein was added to C1q-depleted serum. Figure 38C illustrates the complement-mediated bactericidal activity of each mAb, as detected against serogroup B strain H44 / 76.

[0125] Figures 39A-39C The study describes the application of fHbp to each well of an ELISA plate and the detection of the inhibitory effect of anti-fHbp mAb on fH binding using ELISA. Figure 39A ), and using live bacteria of serogroup B strain H44 / 76, the inhibitory effect of anti-fHbpmAb on fH binding was detected by flow cytometry. Figure 39B and 39C ).

[0126] Figure 40A-40C The binding of fH to the serogroup B H44 / 76 mutant is described, which causes fHbp expression or genetic inactivation of both fHbp and NspA. Figure 40A The binding of the control anti-PorA mAb; Figure 40B and 40C In human serum where IgG has been removed, fH binding occurs.

[0127] Figures 41A-41E The bactericidal activity of human IgG mouse chimeric anti-fHbp mAb was described, and the assay was performed against the NspA-inactivated serogroup B H44 / 76 mutant. Figure 41A , 41B , and 41C: are anti-fhbp mAb JAR 3, JAR 5 and mAb 502, respectively; Figure 41D and 41E These are the control anti-PorA and anti-capsule mAb, respectively.

[0128] Figure 42The bactericidal activity of the anti-NspA antibody against serogroup A strain (Senegal 1 / 99) is described, targeting either fHbp knockout serogroup A strain (top figure) or control anti-PorA mAb P1.9 (bottom figure).

[0129] Figure 43 Figures AC depict the serum anti-fHbp antibody response in wild-type mice immunized with either a recombinant fHbp vaccine or a natural outer membrane vesicle vaccine derived from the fHbp overexpressing or fHbp knockout serogroup B strain H44 / 76 mutant. The immune response to anti-fHbp antibodies was detected using ELISA (Figure A), or the ability of serum anti-fHbp antibodies to inhibit the binding of fH to fHbp was assessed using ELISA (Figures B and C, also detected using ELISA). Mice were immunized with either a recombinant fHbp ID1 vaccine (solid triangle) or a NOMV vaccine derived from the fHbp ID1 overexpressing (hollow circle) or fHbp knockout (solid circle) serogroup B strain H44 / 76 mutant.

[0130] Figure 44 The amino acid sequence represents the surface protein A (NspA) polypeptide (SEQ ID NO:15) of Neisseria genus.

[0131] Figure 45 Amino acid sequences of various naturally occurring factor H binding proteins (fHbp): fHbp ID 1, fHbp ID 15, fHbp ID 22, fHbp ID 28, fHbp ID 77, and chimera I (Beernink et al. (2008) Infec. Immun. 76:2568-2575). The displayed fHbp ID sequences have no leader sequence. In the sequence of chimera I, lowercase letters correspond to amino acid sequences derived from fHbp ID 1, while uppercase letters correspond to amino acids derived from ID 77.

[0132] Before describing the invention and specific exemplary embodiments thereof, it should be understood that the invention is not limited to the specific embodiments described, as these can obviously be varied. It should also be understood that the terminology used in this application is intended only to describe specific embodiments and not to be limiting, as the scope of the invention will be limited only by the appended claims.

[0133] When a numerical range is mentioned, it should be understood that this invention includes every intermediate value between the upper and lower limits of the range, as well as any other value or intermediate value described within the described range, where the intermediate value is as small as one-tenth of the lower limit unit, unless the context otherwise indicates. This invention also includes the upper and lower limits of these smaller ranges that may be independently included within them, except for end values ​​explicitly excluded from the described range. When the described range includes one or two end values, the scope of this invention also includes the range excluding these included end values.

[0134] For clarity, certain features of the invention are described in different embodiments, and it should be understood that these features may also be provided in combination in a single embodiment. Conversely, for brevity, multiple features of the invention are described in a single embodiment, and these features may also be provided individually or in any suitable sub-combination. The invention specifically includes and discloses all combinations of embodiments involving amino acid modifications, including amino acid substitutions, compared to a control amino acid sequence, as if each combination were independently and explicitly disclosed, provided that such combinations contain a polypeptide having the desired characteristics, for example, a non-naturally occurring fHbp polypeptide with an affinity for human fH lower than fHbp ID 1. Furthermore, the invention explicitly includes and discloses all combinations of such amino acid modifications (including amino acid substitutions) listed in those embodiments describing such amino acid modifications, as if each such combination of such amino acid modifications were independently and explicitly disclosed in this application.

[0135] Unless otherwise stated, all technical terms used in this application have the same meaning as understood by one of ordinary skill in the art to which this invention pertains. While the invention may be practiced or tested using any similar or equivalent methods and materials described herein, preferred methods and materials are described below. All publications mentioned herein are incorporated by reference to disclose and describe methods and / or materials associated with the cited publications.

[0136] It must be noted that the singular forms “a,” “an,” and “the” used in this application and the appended claims include plural objects unless the context clearly indicates otherwise. Thus, for example, referring to “an antigen” also includes multiple such antigens, referring to “the protein” also includes one or more proteins, and so on.

[0137] Publications discussed in this application are cited only because they were published prior to the filing date of this application. Nothing in this application should be construed as an admission that the invention did not precede these publications by way of prior invention. Furthermore, the publication dates provided may differ from the actual publication dates and may require separate verification. Invention Details

[0138] This application provides an H factor binding protein and a method of using the protein, which can induce bactericidal antibodies against at least one strain of Neisseria meningitidis.

[0139] definition

[0140] “H factor binding protein” (fHbp), also known in the literature as GNA1870, GNA 1870, ORF2086, LP2086 (lipoprotein 2086), and “741”, refers to a class of Neisseria meningitidis polypeptides. It was found to be a lipoprotein on the surface of bacteria in nature. Based on the variability of the amino acid sequence and immune cross-reactivity (Masignani et al. (2003) J Exp Med 197:789-99), Neisseria meningitidis strains have been subdivided into three fHbp variant groups (referred to as variant 1 (v.1), variant 2 (v.2), and variant (v.3) in some reports (Masignani et al. (2003) J Exp Med 197:789-99), and as families A and B in other reports (see, for example, Fletcher et al. (2004) Infect Immun 72:2088-2100)). According to neisseria.org or pubmlst.org / neisseria / fHbp / , each unique fHbp found in Neisseria meningitidis is assigned an fHbp peptide ID. Because the lengths of variant 2 (v.2) fHbp proteins (from strain 8047, fHbp ID 77) and variant 3 (v.3) fHbp (from strain M1239, fHbp ID 28) differ from MC58 (fHbp ID 1) by -1 and +7 amino acid residues, respectively, the residue numbers used to identify v.2 and v.3 fHbp proteins differ from the numbering based on the actual amino acid sequences of these proteins. Therefore, for example, the leucine residue (L) at position 166 of the v.2 or v.3 fHbp sequence corresponds to residues at positions 165 and 173 of the v.2 and v.3 proteins, respectively.

[0141] The human H factor (“human fH”) used in this application refers to the factor containing Figure 9B The protein with the amino acid sequence shown in (SEQ ID NO: 9) and its naturally occurring human allele variant.

[0142] The terms "heterogeneous" or "chimera" refer to two components defined by structures from different sources or ancestral sequences. For example, when used in the context of a chimeric polypeptide, the chimeric polypeptide comprises operatively linked amino acid sequences derived from different polypeptides of different phylogenetic groups (e.g., a first component derived from an α-ancestral amino acid sequence and a second component derived from a β-ancestral amino acid sequence). A chimeric polypeptide contains two or more defined fragments, each derived from a different ancestor, and can be either naturally occurring or artificially prepared (non-natural) polypeptides. For more details on naturally occurring chimeras, see Beernink PT, Granoff DM (2009) Microbiology 155:2873-83. Non-natural chimeras refer to "artificially prepared chimeras" that contain fHbp with a heterologous component not found in nature.

[0143] "Heterologous" or "chimeric" polypeptides may also contain two or more different components, each derived from a different fHbp (e.g., variant 1, 2, or 3). The components may be operatively linked at any site along the length of the fHbp polypeptide.

[0144] In the context of a polynucleotide encoding any chimeric polypeptide as described above, “heterogeneous” includes operatively linked nucleic acid sequences from different genes (e.g., from a first component of the nucleic acid encoding the fHbp v.1 polypeptide and a second component of the nucleic acid encoding the fHbp v.2 polypeptide) or nucleic acid sequences from different progenitor amino acid sequences (α or β).

[0145] Other exemplary “heterologous” nucleic acids include expression constructs in which a nucleic acid encoding a sequence is operatively linked to a regulatory element (e.g., a promoter) whose genetic origin differs from that of the encoding sequence (e.g., the genetic origin of the host cell may differ from that of the promoter, the encoding sequence, or both, to provide expression in a target host cell). For example, the T7 promoter is referred to as a heterologous nucleic acid when it is operatively linked to a polynucleotide encoding an fHbp polypeptide or a domain thereof.

[0146] In the context of recombinant cells, "heterologous" refers to the presence of nucleic acids (or gene products, such as polypeptides) in the host cell that have a different genetic origin. For example, the amino acid or nucleic acid sequence of one Neisseria strain is heterologous to the host of another Neisseria strain.

[0147] In the context of an amino acid sequence or polynucleotide sequence (e.g., an amino acid sequence “derived from” fHbp ID 1), “derived from” indicates that the sequence of the polypeptide or nucleic acid is based on a control polypeptide or nucleic acid sequence (e.g., a naturally occurring fHbp protein or encoding nucleic acid), and is not intended to limit the source or method of preparing the protein or nucleic acid. Non-limiting examples of control polypeptides and control polynucleotides from which the amino acid sequence or polynucleotide sequence may be “derived” include naturally occurring fHbp, fHbp ID 1, and non-naturally occurring fHbp. In the context of a bacterial strain, “derived from” indicates a strain obtained through in vivo or in vitro passage culture of a parent strain, and / or recombinant cells obtained by modifying a parent strain.

[0148] "Conservative amino acid substitution" refers to the substitution of one amino acid residue on the side chain of an amino acid by another amino acid with similar chemical and physical properties (e.g., charge, size, hydrophobicity / hydrophilicity). "Conservative substitution" aims to include substitutions within the following amino acid residue groups: gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr; lys, arg; and phe, tyr. Such substitutions are guided by aligning the amino acid sequence of the polypeptide containing the epitope of interest.

[0149] The term "protective immunity" refers to the administration of a vaccine or immunization program to mammals to induce an immune response that prevents, delays, reduces the severity of, or reduces or eliminates the symptoms of disease caused by Neisseria meningitidis. Protective immunity may be accompanied by the production of bactericidal antibodies. It should be noted that it is generally accepted in the art that the production of bactericidal antibodies against Neisseria meningitidis indicates that the vaccine has a protective effect in humans (Goldschneider et al. (1969) J.Exp.Med. 129:1307; Borrow et al. (2001) InfectImmun. 69:1568).

[0150] The phrase "illness caused by Neisseria meningitidis strains" includes any clinical symptoms or combinations of clinical symptoms presenting in humans infected with Neisseria meningitidis. These symptoms include, but are not limited to: Neisseria meningitidis pathogens residing in the upper respiratory tract (e.g., nasopharyngeal and tonsillar mucosa), bacterial entry into the mucosal and submucosal vascular beds, sepsis, septic shock, inflammation, hemorrhagic skin lesions, activation of fibrinolysis and blood clotting, organ dysfunction such as renal, pulmonary, and heart failure, adrenal hemorrhage and muscle infarction, capillary leakage, edema, peripheral limb ischemia, acute respiratory distress syndrome, pericarditis, and meningitis.

[0151] In the context of an antigen (e.g., a peptide antigen), the phrase "specifically binds to an antibody" or "a specific immune response" refers to a binding reaction based on and / or testing the presence of an antigen in a sample from a heterogeneous population that may contain other molecules. Therefore, under specified conditions, the one or more specific antibodies can bind to one or more specific antigens in a sample without binding extensively to other molecules present in the sample. Similarly, the phrase "specifically binds to an antibody" or "a specific immune response" refers to a binding reaction based on and / or testing the presence of an epitope (e.g., a peptide epitope) in a heterogeneous population of antigens (e.g., peptides) and in a heterogeneous population that may contain other epitopes. Therefore, under specified conditions, the one or more specific antibodies can bind to a specific epitope in an antigen without binding extensively to the antigen and / or other epitopes present in the sample.

[0152] The phrase "sufficient dosage to elicit an immune response" indicates a detectable difference between indicators of the immune response detected before and after administration of a specific antigen. Indicators of the immune response include, but are not limited to, antibody titers or specificities detected by the following assays: enzyme-linked immunosorbent assay (ELISA), sterilization assays, flow cytometry, immunoprecipitation, Ouchterlony immunodiffusion; binding assays such as dot blot, Western blot, or antigen array assays; cytotoxicity assays, etc.

[0153] "Surface antigens" are antigens present in the surface structures of Neisseria meningitidis (e.g., outer membrane, capsule, pili, etc.).

[0154] "Separated" means that the environment in which the target compound exists is different from that in which it naturally occurs. "Separated" is intended to include compounds in a sample in which the target compound is sufficiently enriched, and / or in which the target compound is partially or sufficiently purified.

[0155] "Enrichment" means performing non-natural manipulations on a sample (e.g., by an experimenter or clinician) to increase the concentration of the target compound to (e.g., at least 3, 4, 8, 64, or more times greater) the concentration of the compound in a starting sample, such as a biological sample (e.g., a sample in which the compound is naturally present or is present after administration) or a sample in which the compound is prepared (e.g., in the case of bacterial peptides, antibodies, chimeric peptides, etc.).

[0156] Target gene "knockout" refers to a gene sequence alteration that leads to a reduction in the function of a target gene, such as causing the target gene expression to be undetectable or insignificant, and / or the gene product to be nonfunctional or lacking significant function. For example, knocking out a gene involved in LPS synthesis can significantly reduce gene function, resulting in undetectable or only insignificant gene expression, and / or significantly reduced or undetectable biological activity (e.g., enzyme activity) of the gene product compared to before modification. "Knockout" includes conditional knockout, such as when the target gene is altered upon exposure to pre-defined conditions (e.g., temperature, osmotic pressure) or upon exposure to substances that promote changes in the target gene. Target gene "knock-in" refers to a genetic alteration of a gene that results in enhanced function of the target gene.

[0157] FHBP peptide with altered FH binding properties

[0158] Before describing the more fHbps to be disclosed in this invention, it is helpful to describe some naturally occurring fHbps. As shown on the websites neisseria.org and pubmlst.org / neisseria / fHbp, each unique and naturally occurring fHbp found in Neisseria meningitidis is assigned an fHbp peptide ID. This fHbp naming convention will be adopted in this invention.

[0159] For convenience and clarity, the natural amino acid sequence of fHbp ID 1 (v.1fHbp of Neisseria meningitidis strain MC58) was selected as the control sequence for all naturally occurring and non-naturally occurring fHbp amino acid sequences in this application, including fHbp chimeras and / or variants described in this application. The amino acid sequence of fHbp ID 1 is shown below. Figure 45 And the following text:

[0160] fHbp ID1

[0161] CSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEK TYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKRQFRIGDIAGEHTSFDKLPEGGRATYRGTAFGSDDAGGKLTYTIDFAA KQGNGKIEHLKSPELNVDLAAADIKPDGKRHAVISGSVLYNQAEKGSYSLGIFGGKAQE VAGSAEVKTVNGIRHIGLAAKQ (SEQ ID NO: 1).

[0162] When referring to the positions of amino acid residues in fHbp, the position numbers used in this application correspond to the amino acid residue numbers of fHbp ID 1. A comparison of different fHbps and the amino acid residues corresponding to fHbp ID 1 is provided below. Figure 19 .like Figure 19 As shown in SEQ ID NO:1, position number 1 refers to the first amino acid residue in fHbp ID 1, which is cysteine. In some cases, the N-terminus of the fHbp involved in this application may contain an additional leader sequence. For example, fHbp ID1 may have the leader sequence MNRTAFCCLSLTTALILTA (SEQ ID NO:16) at its N-terminus. However, in this application, amino acid position number 1 in any fHbp is still defined as the position corresponding to the cysteine ​​residue at amino acid position 1 in fHbp ID 1 as shown above in the alignment, and if a leader sequence is present, position number 1 is the first residue after the leader sequence. See details below. Figure 19 .

[0163] This application provides fHbp, compositions containing fHbp, and methods of using said fHbp and compositions. The fHbp has a lower affinity for human fH than the corresponding control fHbp (e.g., naturally occurring fHbp; or other control fHbp). This is because high-affinity fHbp is more likely to form a complex with fH, and the binding to fH can mask one or more epitopes on the fHbp, preventing them from being recognized by the host immune system. Therefore, fHbp complexed with and / or bound to fH may not function as an effective immunogen as uncomplexed fHbp. Conversely, when fHbp with relatively low affinity for fH is administered as an immunogen (e.g., in a vaccine composition), it can present certain epitopes to the immune system of the immunized host that cannot be presented by fHbp with high affinity for fH because binding to fH may mask such epitopes. The fHbp has a low affinity for human fH and can be used to induce bactericidal antibodies against Neisseria meningitidis and / or provide protective immunity against Neisseria meningitidis. The fHbp is a non-naturally occurring fHbp. Non-naturally occurring fHbp cannot be found in nature; it is artificially prepared and / or intentionally modified. The non-naturally occurring fHbp can be prepared by chemical synthesis or recombinant methods.

[0164] As used in this application, "low affinity," "lower affinity," or "fH low-binding protein" means that the binding affinity of fHbp to human fH is as low as, or lower than, fHbp ID 1. Therefore, the target fHbp includes fHbp ID 14 and fHbp ID 15, because fHbp ID 14 and fHbp ID 15 have a lower affinity for human fH than fHbp ID 1.

[0165] The binding affinity of low-affinity fHbp to human fH is no more than about 100%, no more than about 95%, no more than about 90%, no more than about 85%, no more than about 80%, no more than about 75%, no more than about 70%, no more than about 65%, no more than about 60%, no more than about 50%, no more than about 45%, or lower than the affinity of high-affinity fHbp (e.g., fHbp ID 1) to human fH. For example, the affinity of object fHbp to human fH is less than about 50% of the affinity of fHbp ID 1 to human fH.

[0166] In some embodiments, the binding affinity of the non-naturally occurring fHbp to human fH is 85% or less of the binding affinity of wild-type fHbp to human fH. For example, in some embodiments, the binding affinity of the non-naturally occurring fHbp to human fH is about 85% to about 75%, about 75% to about 65%, about 65% to about 55%, about 55% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%. For example, in some embodiments, the binding affinity of the object's non-naturally occurring fHbp to human fH is about 85% to about 75%, about 75% to about 65%, about 65% to about 55%, about 55% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%.

[0167] For example, in some embodiments, the binding affinity of a mutant of object fHbp ID1 (e.g., an R41S, R41A, R130A, H119A, E218A, or E239A mutant of fHbp ID1) to human fH is about 85% to about 75%, about 75% to about 65%, about 65% to about 55%, about 55% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%.

[0168] For example, in some embodiments, the binding affinity of a mutant of fHbp ID 4, ID 9, or ID 74 (e.g., an R41S mutant of fHbp ID4, ID9, or ID74) to human fH is about 85% to about 75%, about 75% to about 65%, about 65% to about 55%, about 55% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%.

[0169] For example, in some embodiments, the binding affinity of a mutant of object fHbp ID 22 (e.g., R80A, D211A, E218A, E248A, G236I, or T220A / H222A mutant of fHbp ID 22) to human fH is about 85% to about 75%, about 75% to about 65%, about 65% to about 55%, about 55% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%.

[0170] For example, in some embodiments, the binding affinity of a mutant of object fHbp ID 77 (e.g., R41S / K113A, R41S / K119A, R41S / D121A, or R41S / K113A / D121A mutant of fHbp ID 77) to human fH is about 85% to about 75%, about 75% to about 65%, about 65% to about 55%, about 55% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%.

[0171] For example, in some embodiments, the binding affinity of a mutant of object fHbp ID 28 (e.g., the E218A mutant of fHbp ID 28; the K199A mutant of fHbp ID 28) to human fH is about 85% to about 75%, about 75% to about 65%, about 65% to about 55%, about 55% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%.

[0172] The term can be used to describe the dissociation constant (K). D This describes the binding affinity. The dissociation constant (Ka) between low-affinity fHbp and human fH is... D ;M) at least higher than about 80%, at least higher than about 100%, at least higher than about 120%, at least higher than about 140%, at least higher than about 160%, at least higher than about 200%, or higher than high affinity fHbp (e.g., fHbp ID 1) with human fH K D It is also possible to use K with low affinity fHbp. D K described as fHbp ID 1 D Approximately 2X, approximately 3X, approximately 5X, approximately 10X, approximately 15X, approximately 20X, up to approximately 50X or more.

[0173] As used in this application, "affinity with human fH is lower than that of the corresponding fHbp" is used to describe fHbp with a binding affinity lower than that of the corresponding control fHbp.

[0174] In many cases, the corresponding fHbp used to compare the binding affinity of the target fHbp (“control f'Hbp”) is fHbpID 1. Other corresponding fHbp that can serve as typical controls include variant 2 fHbp (e.g., fHbp ID 22 or 77), variant 3 (e.g., fHbp ID 28) (Masignani et al. (2003) J Exp Med 197:789-99 and Pajon R et al. (2010) Vaccine 28:2122-9), other variant 1 fHbp (e.g., fHbp ID 4, 9, or 94), naturally occurring chimeras, or artificially prepared chimeric fHbp.

[0175] The amino acid sequences of some naturally occurring fHbp and artificially prepared chimeras are shown below, and see also [link to relevant documentation]. Figure 45 .

[0176] FHbp ID 22

[0177] CSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYG NGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPSGKAEYHGKAFSSDDPNGRLHYSIDFTKKQGYG RIEHLKTPEQNVELASAELKADEKSHAVILGDTRYGGEEKGTYHLALFGDRAQEIAGSA TVKIREKVHEIGIAGKQ(SEQ ID NO:2)

[0178] FHbp ID 28

[0179] CSSGGGGSGGGGVAADIGGTGLADALTAPLDHKDKGLKSLTLEDSIPQNGTLTLSAQGAE KTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQNHSAVVALQIEKINNPDKTDSLINQRSFLVSGLGGEHTAFNQLPGGKAEYHGKAFSSDDPNGRLHYSIDF TKKQGYGRIEHLKTLEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ(SEQ ID NO:3)

[0180] FHbp ID 77

[0181] CSSGGGGVAADIGARLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYG NGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPD KIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGS ATVKIGEKVHEIGIAGKQ(SEQ ID NO:4)

[0182] FHbp ID 15

[0183] CSSGGGGSGGGGVAADIGAGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQ GAERTFKAGDKDNSLNTGKLKNDKISRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQTEQVQDSEHSGKMVAKRQFRIGDIVGEHTSFGKLPKDVMATYRGTAFGSDDAGGKL TYTIDFAAKQGHGKIEHLKSPELNVDLAAADIKPDEKHHAVISGSVLYNQAEKGSYSLGIFGGQAQEVAGSAEVETANGIRHIGLAAKQ(SEQ ID NO:5)

[0184] FHbp ID 6

[0185] CSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEK TYGNGDSLNTGKLKNDKVSRFDFIRQIEVNGQLITLESGEFQVYKQSHSALTALQTEQV QDSEHSRKMVAKRQFRIGDIAGEHTSFDKLPKGDSATYRGTAFGSDDAGGKLTYTIDFAAKQGYGKIEHLKSPELNVDLAAAYIKPDEKHHAVISGSVLYNQDEKGSYSLGIFGGQAQ EVAGSAEVKTANGIRHIGLAAKQ(SEQ ID NO:6)

[0186] FHbp ID 14

[0187] CSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEK TYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQTEQEQDPEHSGKMVAKRRFKIGDIAGEHTSFDKLPKDVMATYRGTAFGSDDAGGKLTYTIDFA AKQGHGKIEHLKSPELNVELATAYIKPDEKHHAVISGSVLYNQDEKGSYSLGIFGGQAQ EVAGSAEVETANGIHHIGLAAKQ(SEQ ID NO:7)

[0188] Chimera I

[0189] cssggggvaadigagladaltapldhkdkglqsltldqsvrkneklklaaqgaektygngdslntgklkndkvsrfdfirqievdg qlitlesgefqvykqshsaltafqteqiqdsehsgkmvakrqfrigdiaGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTY HLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ(SEQ ID NO:8)(Beernink et al. (2008) Infec. Immun. 76:2568-2575). For example Figure 45As shown, lowercase letters correspond to amino acid sequences derived from fHbp ID 1, while uppercase letters correspond to amino acids derived from fHbp ID 77. The position corresponding to R41 in fHbp ID 1 is represented by the bold lowercase letter "r".

[0190] The corresponding fHbp can be naturally occurring and / or non-natural (e.g., artificially prepared chimeric) fHbp from which the target fHbp is derived. Naturally occurring chimeric fHbp have variable segments derived from different ancestral (α or β). Due to these variable segments, the molecular architecture appears modular, and fHbp variants can be further classified into modular groups based on different combinations of the five variable segments, each variant derived from one of two genetic lineages named α- or β-type (Pajon R et al. (2010) Vaccine 28:2122-9; Beernink PT, Granoff DM (2009) Microbiology 155:2873-83). The six modular groups named I through VI can cover >95% of all known fHbp variants (Pajon R et al. (2010) Vaccine 28:2122-9). See the architecture of the naturally occurring fHbp modular groups for details. Figure 16 .

[0191] The corresponding fHbp can be an fHbp in a fragment (e.g., a variable fragment defined in a modular architecture) or a full-length mature protein that has a high amino acid sequence identity with the target fHbp (e.g., having at least about 99%, at least about 95%, at least about 90%, at least about 85%, at least about 80%, or at least about 75% amino acid sequence identity).

[0192] The corresponding fHbp used as a control in the binding affinity comparison of the object fHbp may also include, in one or more segments, an fHbp that shares the same ancestor (α or β) as the corresponding segment of the object fHbp.

[0193] The target fHbp may contain an amino acid sequence that has at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% amino acid sequence identity with the control fHbp; and differ from the control fHbp by one to ten amino acids, for example, by one, two, three, four, five, six, seven, eight, nine, or ten amino acids. Therefore, the target fHbp may have at most one, two, three, four, or up to ten modifications (e.g., substitution, deletion, or insertion) relative to a naturally occurring and / or non-natural (e.g., chimeric) fHbp from which it is derived. One or more amino acid alterations may reduce the affinity of the fHbp for human fH compared to the unaltered fHbp. As shown above, the fHbp from which the object fHbp is derived includes naturally occurring fHbp and non-naturally occurring fHbp. Non-naturally occurring fHbp may include artificially prepared chimeras, such as chimeras known in the art and chimeras described in PCT application number WO2009 / 114485, the disclosure of which is incorporated herein by reference.

[0194] Therefore, in some embodiments, the target fHbp contains a single amino acid substitution relative to the control fHbp (e.g., where the control fHbp is a naturally occurring fHbp (e.g., fHbp ID1, or an artificially prepared chimera)). In some embodiments, the target fHbp contains a single amino acid substitution (i.e., only one amino acid substitution) relative to a naturally occurring fHbp (e.g., fHbp ID 6, fHbp ID 14, fHbp ID 15, fHbp ID 22, fHbp ID 28, fHbp ID 77, or other naturally occurring fHbp). The amino acid sequences of fHbp ID 1, fHbp ID 15, fHbp ID 22, fHbp ID 28, and fHbp ID 77 are as follows: Figure 19 and Figure 45As shown; the amino acid sequences of fHbp ID 6 and fHbp ID 14 are as described above. In some embodiments, the target fHbp contains a single amino acid substitution relative to fHbp ID 1 (i.e., only one amino acid substitution). In some embodiments, the target fHbp contains 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions relative to the control fHbp. In some embodiments, the target fHbp contains 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions relative to fHbp ID 1. In some embodiments, the target fHbp contains substitutions relative to naturally occurring fHbp (e.g., fHbp ID 6, fHbp ID 14, fHbp ID 15, fHbp ID 28, etc.). Figure 19 The target fHbp may contain 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions as shown or otherwise naturally occurring. In some embodiments, the target fHbp contains 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions relative to one of the amino acid sequences in fHbp ID 1, fHbp ID 15, fHbp ID 22, fHbp ID 28, and fHbp ID 77.

[0195] By comparing the amino acid sequences of low-binding fH proteins (e.g., fHbp ID 14 and / or fHbp ID 15) with the amino acid sequences of fHbps with affinity for human fH equivalent to, for example, fHbp ID 1, the location of the introduced amino acid residue change can be determined. In this application, fHbps with affinity for human fH equivalent to or higher than fHbp ID 1 are referred to as "high-binding fH proteins." Some examples of high-binding fH proteins include fHbp ID 1, fHbp ID 6, and fHbp ID 28. Low-binding and high-binding fH proteins sharing one or more ancestral fragments can be compared during sequence alignment. Figure 19 This is an example of sequence alignment, which is used to determine which amino acid changes can be made to a naturally occurring fHbp to obtain the target fHbp.

[0196] Variants of the target fHbp can be derived from variant 1 fHbp, variant 2 fHbp, or variant 3 fHbp (e.g., they may contain one or more amino acid substitutions compared to these variants). Variants of the target fHbp can be derived from module group I fHbp, module group IIfHbp, module group III fHbp, module group IV fHbp, module group V fHbp, module group VI fHbp, module group VII fHbp, module group VIII fHbp, module group IX fHbp, or module group X fHbp (e.g., they may contain one or more amino acid substitutions compared to these variants).

[0197] Compared to the control fHbp, amino acid substitutions that may reduce the affinity of fHbp for fH include: amino acid substitutions at contact residues in the fHbp amino acid that bind to fH; amino acid substitutions exposed on the surface of the fHbp amino acid; amino acid substitutions at the interface between the amino-terminal and carboxyl-terminal domains in the fHbp amino acid; and amino acid substitutions adjacent to the fH-binding residues in the fHbp amino acid, wherein an amino acid "adjacent" to the fH-binding amino acid is defined as an amino acid that is one to ten residues away from the amino-terminus or carboxyl-terminus of the fH-binding amino acid. In some embodiments, the amino acid substitutions that reduce the affinity for fH are not fH contact residues. Some contact residues are described below. Figure 19 The bolded part in the text.

[0198] When fHbp contains amino acid substitutions relative to naturally occurring or relative to fHbp ID 1, the amino acid substitutions may be conserved relative to said amino acid substitution. For example, if R41 is modified to S, resulting in an R41S substitution, and in particular the R41S substitution reduces its affinity for human fH, the disclosure of this application also includes conserved amino acid substitutions relative to S, thereby also including amino acid substitutions of R41T.

[0199] This application provides for non-naturally occurring fHbp with an amino acid substitution at position 41 relative to fHbp ID1, wherein the amino acid substitution structurally interferes with the interaction between fHbp and human H factor, provided that the fHbp mutant retains immunogenicity. Suitable amino acids for substitution at position 41 relative to fHbp ID1 include hydrophobic residues (e.g., Gly, Ala, Val, Leu, Ile, Pro); small polar residues (e.g., Ser, Cys, Thr, Met, Asn, Gln); small charged residues (e.g., Asp, Glu); and large hydrophobic residues (e.g., Phe, Trp). Certain substitutions are not expected to significantly interfere with the interaction between fHbp and human H factor and should therefore be avoided; examples of such substitutions include large charged residues (e.g., Lys).

[0200] In some embodiments, amino acid substitutions of one or more of the following residues are specifically excluded: R41, Q38, Q87, Q113, K113, K119, D121, G121, Q126, Q128, R130, D201, E202, E218, A235, E239, and K241. In some embodiments, for example, when the target fHbp contains a single amino acid substitution relative to the control fHbp (e.g., when the control fHbp is a naturally occurring fHbp or fHbp ID 1), the single amino acid substitution may be located at position E218 or E239.

[0201] The amino acid changes in object fHbp include, Figure 19 Residues highlighted in shading and / or in boxes are listed below. In one example of sequence analysis, fragment A(V) of the low-binding protein fHbp ID 15 was compared. A (Residues 8-73) share the same ancestral sequence as the fH-high binding protein fHbp ID 28. A Thus, except for positions 41 and 60, the V values ​​of fHbp ID 15 and fHbp ID 28 are... A The fragments have the same amino acid sequence. For example... Figure 19 As shown, the V of fHbp 15 A V with S41 and R60; fHbp ID 28 A It contains P41 and K60. Based on this analysis, the amino acid residue positions corresponding to positions 41 and 60 of fHbp ID 15 were considered as candidate positions for obtaining target fHbp by introducing alterations. In other words, control fHbp that do not have S and R at residue positions corresponding to positions 41 and 60 of fHbp 15, respectively, can be mutated to have S and / or R at positions 41 and 60 of fHbp 15. Target fHbp containing one or more amino acid substitutions may have a lower affinity for human fH than unsubstituted fHbp (e.g., S41P, S41A, R41P, or R41A). Such fHbps are included in the target fHbp, which serve as immunogens in the desired target to induce bactericidal antibodies.

[0202] Other candidate residue positions where amino acid alterations can be introduced are discussed in the examples below. For example, the fHbp of the present invention may have amino acid substitutions at one or more positions corresponding to one or more amino acid residues selected from fHbp ID 1, namely 41, 60, 114, 113, 117, 119, 121, 128, 130, 147, 148, 149, 178, 195, 218, 239, 241, or 247 (e.g., based on the numbering of mature fHbp). The fHbp of the present invention may have amino acid substitutions at one or more positions, the positions corresponding to one or more amino acid residues selected from fHbp ID 1, namely 41, 60, 80, 113, 114, 117, 119, 121, 128, 130, 147, 148, 149, 178, 195, 199, 211, 220, 222, 236, 241, 247, or 248, based on the numbering of mature fHbp. The fHbp of the present invention may have amino acid substitutions at one or more positions, the positions corresponding to one or more amino acid residues selected from fHbp ID 1, namely 87, 109, 115, 118, 126, 138, 197, 201, 202, 203, 209, 217, 225, 235, or 245, based on the numbering of mature fHbp. When the corresponding fHbp is variant 2 or variant 3 fHbp (or their respective module groups), modifications may be introduced at positions 113, 119, and / or 121, or any combination thereof. For example, variant 2 fHbp (e.g., fHbp ID 77) may contain substitutions at one or more positions 113, 119, and / or 121, and a serine substitution at position 41, or other suitable substitutions described above at position 41. When the corresponding fHbp is variant ID 22, modifications may be introduced at positions 80, 211, 218, 220, 222, 236, or 248, or any combination thereof.

[0203] The variant H factor binding protein (fHbp) of the present invention may also have amino acid substitutions at one or more positions, said positions corresponding to one or more amino acid residues selected from fHbp ID 1, namely 60, 114, 117, 147, 148, 149, 178, 195, or 247. This is in contrast to the V of fHbp ID 15 discussed above. A Similarly, other locations can be determined by sequence alignment studies between fH low-binding proteins and fH high-binding proteins.

[0204] The variant fHbp of the present invention may be a variant of fHbp ID 1 and may include one, two, three, or four of the following substitutions: R41S, R41A, R130A, H119A, E218A, and E239A. As discussed above, the variant fHbp of the present invention may include a single amino acid substitution. The variant fHbp of the present invention may also include a double amino acid substitution. For example, the variant fHbp of the present invention may include two substitutions selected from R41S, R41A, R130A, H119A, E218A, and E239A.

[0205] The variant fHbp of the present invention may also have amino acid substitutions at one or more positions, the positions corresponding to one or more amino acid residues selected from fHbp ID 1, namely 80, 211, 218, 220, 222, 236, and 248. The corresponding positions in the fHbp variants are readily determined, for example by... Figure 19 , 34 The comparison in 35 and 36 determines the presence of amino acids. As a non-limiting example, the variant fHbp of the present invention may be a variant of fHbp ID 22 and may include one, two, three, or four of the following substitutions: R80A, D211A, E218A, T220A, H222A, G236I, and E248A. As discussed above, the variant fHbp disclosed in this invention may include a single amino acid substitution. The variant fHbp of the present invention may also include a double amino acid substitution. For example, the variant fHbp of the present invention may include substitutions at two positions in R80, D211, E218, T220, H222, G236, and E248. As a non-limiting example, the variant fHbp may include a T220A / H222A double substitution.

[0206] The variant fHbp of the present invention may have amino acid substitutions at one or more positions, the positions corresponding to one or more amino acid residues selected from fHbp ID 77, namely 41, 113, 119, and 121. As a non-limiting example, the variant fHbp of the present invention may be a variant of fHbp ID 77 and may include one, two, three, or four of the following substitutions: R41S, K113A, K119A, and D121A. As discussed above, the variant fHbp of the present invention may include a single amino acid substitution. The variant fHbp of the present invention may also include a double amino acid substitution. For example, the variant fHbp of the present invention may include a substitution of two of R41S, K113A, K119A, and D121A. As a non-limiting example, the variant fHbp may include a double substitution of R41S / K113A, a double substitution of R41S / K119A, or a double substitution of R41S / D121A.

[0207] The variant fHbp of the present invention may have amino acid substitutions at one or more positions, the positions corresponding to one or more amino acid residues selected from fHbp ID 28, namely 113, 121, 199, and 218.

[0208] When position 41 of fHbp in this invention is replaced by serine, the corresponding fHbp belongs to Figure 16 One of the module groups shown. For example, the corresponding fHbp may come from module group I, where all variable segments are of the α lineage. Examples of such object fHbp include the R41S mutants of fHbp IDs 1, 4, 9, and 94. In some embodiments, object fHbp does not include mutants that do not have a reduced affinity for human fH relative to their corresponding fHbp. For example, object fHbp does not include the R41S mutants of fHbp IDs 19 and 22.

[0209] chimeric fHbp

[0210] As shown above, naturally occurring fHbp or artificially prepared fHbp (e.g., artificially prepared chimeric fHbp) can be modified with one or more modifications. Modifications may include modifications in a fragment or a domain, while other fragments and / or domains may be derived from any fHbp (e.g., naturally occurring fHbp from different variant groups).

[0211] The already described modular architecture V A V B V C V D 、 and V E In the fHbp of the fragment, modifications can be introduced into the V of the α spectrum. A In (e.g., introducing R41S in VA of fHbp ID 1), while other segments of fHbp (e.g., V) B V C V D 、 and V E Each can be independently derived from any lineage, any variant group, or any fHbp ID. In another example, the V of object fHbp... A V C 、 and V E The fragment can be derived from the α lineage (lineage 1), while V B and V D It can be derived from the β lineage. When the modification is performed by replacing arginine at position 41 with serine, the modification is introduced into the α-ancestor V. A (V A At position α). The V AA fragment is a continuous amino acid sequence that begins at residue position 7 and ends at residue position 73, and its position number corresponds to the position number of the control sequence fHbp ID 1.

[0212] The fHbp of the present invention can be used in V A The α fragment contains the R41S mutation, the V A The α fragment contains an amino acid sequence that has at least about 90%, at least about 92%, at least about 94%, at least about 95%, at least about 96%, at least about 98%, at least about 99%, up to 100% identity with the following sequences:

[0213] V A The α sequence is listed here, and the R41S mutation is indicated in bold. Therefore, fHbp containing the R41S modification is in V A The α segment contains an R41S mutation and can have a V mutation. B V C V D 、 and V E The fragments are each independently derived from any other fHbp (e.g., different lineages, different variant groups, or mutants of fHbp).

[0214] The chimeric fHbp of the present invention can also be described as having a modification in the N-terminal domain (fHbpN) of fHbp, while the C-terminal domain (fHbpC) can be derived from different fHbp (e.g., different variant groups or different lineages). “fHbpN” refers to a continuous amino acid sequence starting at approximately residue position 8 and ending at approximately residue position 136. “fHbpC” refers to a continuous amino acid sequence starting at approximately residue position 141 and ending at approximately residue position 255. The insertion sequence between fHbpN and fHbpC is a linker between the two domains. For example, the fHbpN of the target fHbp may contain an R41S mutation in a sequence derived from fHbp ID 1, while fHbpC is derived from variant 2 or variant 3 fHbp (e.g., fHbp ID 77).

[0215] The corresponding chimeric fHbp can be any known artificially prepared chimera, such as those described in Beernink et al. (2008) Infec. Immun. 76:2568-2575 and WO 2009 / 114485, the disclosure of which is incorporated herein by reference. The modified chimera exhibits reduced affinity for human fH relative to the chimeric fHbp, but retains epitopes that play a crucial role in evoking a bactericidal response, such as those found in the corresponding chimeric fHbp. fHbp epitopes that can be retained in the modified chimera include those found in the corresponding chimeric fHbp, such as those described in WO 2009 / 114485, the disclosure of which is incorporated herein by reference. For example, the modified chimeric fHbp may contain epitopes that play a crucial role in evoking a bactericidal response against strains containing variant 1 fHbp (e.g., epitopes in the N-terminal domain, such as epitopes defined in mAb JAR 4 and / or JAR 5) and / or strains containing variant 2 or 3 fHbp (e.g., epitopes defined in mAb JAR 10, JAR 11, JAR 13, and / or JAR 36). For example, as Figure 19 and Figure 45 As shown, the R41S mutation can introduce chimeric fHbp modification to reduce binding to human fH, but still retains JAR4 and JAR5 epitopes.

[0216] A characteristic of the target fHbp is that, when administered to a host (e.g., a mammal such as a mouse or human), the target fHbp can elicit a bactericidal response at a level comparable to or slightly higher than that elicited by fHbp ID 1 or other corresponding controls (e.g., fHbp ID 4, 9, 22, 28, 74, or 77). Methods for determining the level of bactericidal response are well known in the art, as described in the Examples section below. For example, the geometric mean of the bactericidal titer in mice immunized with the target fHbp is at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 150%, at least about 175%, at least about 200%, or more than 200% of the geometric mean of the bactericidal titer in mice immunized with fHbp ID 1. In some cases, the geometric mean of bactericidal titers in mice immunized with target fHbp was at least 2, at least 2.5, at least 5, at least 10, or more than 10 times higher than the geometric mean of bactericidal titers in control mice immunized with fHbp ID1.

[0217] The target fHbp excludes those that elicit a bactericidal response significantly lower than fHbp ID 1. The target fHbp excludes fHbp with mutations at residue positions 218 and 239 (e.g., the E218A / E239A double mutant). In some embodiments, the target fHbp includes only non-naturally occurring fHbp; therefore, in some embodiments, the target fHbp excludes naturally occurring fHbp.

[0218] In many cases, target fHbp variants retain and possess conformational epitopes that bind to bactericidal antibodies that are bactericidal against one or more Neisseria meningitidis strains. Therefore, such fHbp mutants can retain epitopes found in naturally occurring fHbp, and their binding affinity to fH is reduced compared to the binding affinity of naturally occurring fHbp to fH. Variants that have minimal or no effect on the conformation of fHbp, thus enabling mutant vaccines to elicit bactericidal antibodies, are considered good vaccine candidates. Whether a variant affects the conformation of fHbp can be determined through various methods, including antibody binding listed in Table 9.

[0219] The fHbp of the present invention may have other features, as detailed below.

[0220] Coupled

[0221] The target fHbp of the present invention may contain one or more additional elements, such as the polypeptide (e.g., having an amino acid sequence heterologous to the target fHbp) and / or a carrier molecule, at the N- and / or C-terminus of the polypeptide. Additional heterologous amino acid sequences may be fused, for example, as a result of expression in bacterial host cells (e.g., *E. coli*) to provide an N-terminal methionine or a derivative thereof (e.g., pyroglutamate), and / or to provide a chimeric polypeptide having a fusion partner at its N- or C-terminus. Target fusion partners include, for example, glutathione S-transferase (GST), maltose-binding protein (MBP), His6-tags, etc., and leader peptides from other proteins, particularly lipoproteins. Fusion partners may provide additional features, such as facilitating the isolation, purification, detection, and immunogenicity of the target fHbp.

[0222] Other elements that can be linked to the target fHbp include carrier molecules (e.g., carrier proteins, such as keyhole hemocyanin (KLH)). Other elements can be linked to peptides via linkers, such as flexible linkers. The carrier contains an immunomodulator, a molecule that directly or indirectly modifies an immune response. A specific class of immunomodulators includes those that stimulate or co-stimulate an immune response. Examples include antigens and antigen carriers, such as toxins or derivatives thereof, including tetanus toxin. Other carriers are also included that facilitate administration and / or enhance immunogenicity in subjects immunized or treated against Neisseria meningitidis. Carrier molecules can also facilitate delivery to target cells or tissues. Additional portions may also contribute to immunogenicity or form complexes with components in vaccines. Carrier molecules may act as scaffold proteins to facilitate the presentation of membrane surface antigens (e.g., vesicle vaccines).

[0223] In one example, the object fHbp is modified at its N- and / or C-terminus to introduce a fatty acid (e.g., an aliphatic carboxylic acid group). The fatty acid can be covalently linked to the fHbp via a flexible linker. An example of a fatty acid that can be used to modify the ends of the object fHbp (e.g., the N-terminus, for example, at the N-terminus) is lauric acid. When covalently linked to other molecules, lauric acid refers to a lauroyl group (e.g., lauroyl sulfate). Lauric acid contains twelve carbon atoms, has ten methylene groups, and has the molecular formula CH3-(CH2). 10 -COOH. Other fatty acids that can be linked to the target peptide include caprylic acid (10 C), myristic acid (14 C), and palmitic acid (16 C). For details, see Westerink MA et al. (1995) Proc.Natl.Acad.Sci.USA 92:4021-4025. It is also contemplated that this application may include any hydrophobic portion capable of anchoring the target fHbp to the bacterial outer membrane, which can be used to couple to the N- and / or C-terminus (e.g., at the N-terminus) of the fHbp of the present invention, wherein said hydrophobic portion may be operatively coupled to the peptide by a linker, such as a flexible linker as described in this application. For example, the hydrophobic pentapeptide FLAV (SEQ ID NO:18), as described in Lowell GH et al. (1988) J.Exp.Med.167:658-63.

[0224] As described above, one method of linking fatty acids and other additional elements described above to fHbp is via a linker (e.g., lauroyl-Gly-Gly). Linkers suitable for modifying the fHbp of the present invention include "flexible linkers". Suitable linkers can be readily selected, and linkers can be of any suitable length, for example, from 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0225] Examples of flexible linkers include glycine polymers (G). n Glycine-serine polymers (including, for example, (GS)) n GSGGS n (SEQ ID NO:19) and GGGS n (SEQ ID NO:20), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are preferred because these amino acids are relatively unstructured and can therefore serve as neutral links between components. Glycine polymers are particularly preferred because, compared to flat alanine, glycine can enter spaces with significantly larger Ramachandran (or phi-psi) spaces and has less restriction compared to residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary flexible linkers include, but are not limited to, GGSG (SEQ ID NO:21), GGSGG (SEQ ID NO:22), GGSG (SEQ ID NO:23), GGSGG (SEQ ID NO:24), GGGSG (SEQ ID NO:25), GSSSG (SEQ ID NO:26), etc. Those skilled in the art will recognize that the design of the fHbp coupled to any of the elements described above can include all or part of a flexible connector, such that the connector can include a flexible connector as well as one or more portions having a less flexible structure.

[0226] Natural fHbp typically contains an N-terminal cysteine ​​residue to which a lipid group may be covalently attached. This cysteine ​​residue is typically lipidated in naturally occurring proteins, and may be lipidated in the fHbp of this invention. Therefore, in the amino acid sequence described herein, the reference to "cysteine" or "C" at that particular position includes both unmodified cysteine ​​and lipidated cysteine ​​(e.g., due to post-translational modification). Thus, the target fHbp may be lipidated or non-lipotropic. Methods for producing lipidated proteins in vitro (see, for example, Andersson et al. (2001) J. Immunological Methods 255:135-48) or in vivo are well known in the art. For example, lipidated fHbp has been purified from membrane components of E. coli proteins by detergent extraction (Fletcher et al. (2004) Infection and Immunity 72:2088-100), a method that may be suitable for producing lipidated fHbp. Lipidified proteins may be preferred because they have higher immunogenicity compared to soluble proteins (see, for example, Fletcher et al. (2004) Infection and Immunity 72:2088-100).

[0227] Understandably, the nucleotide sequence encoding heterologous fHbp can be modified to optimize the codons used, thereby promoting expression in target host cells (e.g., Escherichia coli, Neisseria meningitidis, humans (in the case of DNA-based vaccines), etc.). Methods for producing optimized codon sequences are well known in the art.

[0228] Nucleic acid encoding fHbp

[0229] This application provides a nucleic acid encoding an object fHbp. In some embodiments, the object nucleic acid is present in a recombinant expression construct. A genetically modified host cell comprising the object nucleic acid is also provided.

[0230] The fHbp polypeptide and encoding nucleic acid of this invention can be derived from any suitable Neisseria meningitidis strain. As is well known in the art, Neisseria meningitidis strains are classified into serogroups (capsular groups), serotypes (PorB phenotype), and subtypes (PorA phenotype) based on the reaction of polyclonal antibodies (Frasch, CE and Chapman, 1973, J. Infect. Dis. 127:149-154) or monoclonal antibodies that interact with different surface antigens. Capsular grouping is usually based on immunologically detectable differences in capsular polysaccharides, but this detection method is being replaced by PCR encoding genes for specific enzymes responsible for the biosynthesis of structurally different capsular polysaccharides. Approximately 12 capsular groups are known (including A, B, C, X, Y, Z, 29-E, and W-135). Strains of capsular groups A, B, C, Y, and W-135 can cause almost all meningococcal diseases. Serotypes are conventionally distinguished by antigenic differences in an outer membrane protein called channel protein B (PorB), as determined by monoclonal antibodies. Currently known antibodies can identify approximately 21 serotypes (Sacchi et al., 1998, Clin. Diag. Lab. Immunol. 5:348). Serotypes have also been distinguished by antigenic differences in an outer membrane protein called channel protein A (PorA) determined by antibodies. Serotyping and serotype identification are being replaced by PCR and / or DNA sequencing, which respectively identify genes encoding the variable regions of PorB and PorA related to mAb reactivity (e.g., Sacchi, Lemos et al., ibid.; Urwin et al., 1998, Epidem. and Infect. 120:257).

[0231] Although any capsular group of Neisseria meningitidis strains can be used, Neisseria meningitidis strains with capsular group B are particularly preferred as the source of nucleic acid encoding fHbp and the source of its domain derivation.

[0232] Nucleic acids encoding fHbp peptides can be used to construct the object fHbp included in this application, and these nucleic acids are well known in the art. Different fHbps and their sequences are available at neisseria.org and pubmlst.org / neisseria / fHbp. Examples of fHbp peptides can also be found, for example, US Patent Application No. 61 / 174,424, PCT Application No. PCT / US09 / 36577, WO 2004 / 048404; Masignani et al. (2003) J Exp Med 197:789-799; Fletcher et al. (2004) Infect Immun 72:2088-2100; Welsch et al. J Immunol 2004 172:5606-5615; and WO 99 / 57280. Nucleic acid (and amino acid sequences) of fHbp variants and subvariants are also available in GenBank, accession number: NC_003112, GeneID:904318 (NCBI Ref. NP_274866), fHbpID 1 from Neisseria meningitidis strain MC58; AY548371 (AAT01290.1) (from Neisseria meningitidis strain CU385); AY548370 (AAT01289.1) (from Neisseria meningitidis strain H44 / 76); AY548377 (AAS56920.1) fHbp ID 4. From Neisseria meningitidis strain M4105; AY548376 (AAS56919.1) (from Neisseria meningitidis strain M1390); AY548375 (AAS56918.1) (from Neisseria meningitidis strain NZ98 / 254); AY548374 (AAS56917.1) (from Neisseria meningitidis strain M6190); AY548373 (AAS56916.1) (from Neisseria meningitidis strain 4243); and AY548372 (AAS56915.1) (from Neisseria meningitidis strain BZ83).

[0233] To identify the relevant amino acid sequence intended for use in the object fHbp of this invention, it should be noted that the immature fHbp includes a leader sequence of approximately 19 residues. Furthermore, when an amino acid sequence is provided, those skilled in the art will readily conceive of nucleic acid sequences capable of encoding and expressing a polypeptide having this amino acid sequence.

[0234] In addition to the specific amino acid and nucleic acid sequences provided in this application, the present invention also covers polypeptide and nucleic acid sequences having at least 80%, at least 85%, at least 90%, or at least 95% identity with these examples of polypeptide and nucleic acid sequences. In the context of two or more polynucleotide sequences or two or more amino acid sequences, the term "identity" or "percentage of similarity" refers to a range within a specified region, for example, V... E Alternatively, the comparison may involve amino acid or nucleotide sequences of length at least about 40, 45, 50, 55, 60, 65, or more, up to the full length of the amino acid or nucleotide region (e.g., full-length fHbp), where, when compared or aligned to the maximum correspondence, two or more sequences or subsequences are identical, or have a specific percentage of identical amino acid residues or nucleotides (e.g., at least 80%, at least 85%, at least 90%, or at least 95% identical within a specific region). This invention particularly includes naturally occurring amino acid sequences in various forms and synthetically produced sequences and their encoded nucleic acids.

[0235] When performing sequence comparisons, a control sequence is typically used (e.g., a naturally occurring fHbp peptide sequence or a fragment thereof) and compared to the test sequence. When using a sequence comparison algorithm, the test and control sequences are input into a computer program. If necessary, sequence coordinates are set, and the parameters of the sequence comparison algorithm program are configured. The algorithm then calculates the percentage sequence identity between the test and control sequences based on the set program parameters.

[0236] Examples of algorithms suitable for determining the percentage of sequence identity are BLAST and BLAST 2.0, described by Altschul et al. (1990) J.Mol.Biol.215:403-410 and Altschul et al. (1977) Nucleic Acids Res.25:3389-3402, respectively. Software for BLAST analysis is available to the public from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov). Further exemplary algorithms include ClustalW (Higgins D., et al. (1994) Nucleic Acids Res 22:4673-4680), available at www.ebi.ac.uk / Tools / clustalw / index.html.

[0237] The different positions of certain residues distinguish conserved amino acids. Conserved amino acid substitution refers to the interchange of residues with similar side chains. For example, a group of amino acids with aliphatic side chains includes glycine, alanine, valine, leucine, and isoleucine; a group of amino acids with aliphatic-hydroxyl side chains includes serine and threonine; a group of amino acids with amide side chains includes asparagine and glutamine; a group of amino acids with aromatic side chains includes phenylalanine, tyrosine, and tryptophan; a group of amino acids with acidic side chains includes aspartic acid and glutamic acid; a group of amino acids with base side chains includes lysine, arginine, and histidine; and a group of amino acids with sulfur-containing side chains includes cysteine ​​and methionine.

[0238] Sequence identity between two nucleic acids can also be described using hybridization between the two molecules under stringent conditions. Hybridization conditions are selected from the following standard methods in the art (see, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd edition, (1989) Cold Spring Harbor, NY). An example of stringent hybridization conditions is hybridization at 50°C or above and with 0.1×SSC (15 mM sodium chloride / 1.5 mM sodium citrate). Another example of stringent hybridization conditions is overnight incubation at 42°C in the following solution: 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 mg / ml denatured and cleaved salmon sperm DNA, followed by washing with a 0.1×SSC filter at approximately 65°C. Strict hybridization conditions are hybridization conditions with a degree of strictness at least the same as the representative conditions mentioned above, wherein a condition is considered to be at least strict when it is at least about 80% of the specific strict conditions mentioned above, and typically at least 90%.

[0239] Production methods

[0240] The fHbp of this invention can be produced by any suitable method, including recombinant and non-recombinant methods (e.g., chemical synthesis). When producing the target fHbp using recombinant technology, the method can involve any suitable construct and any suitable host cell, which can be a prokaryotic or eukaryotic cell, commonly a bacterial or yeast host cell, and more commonly a bacterial cell. Methods for introducing genetic material into the host cell include, for example, transformation, electroporation, coupling, and calcium phosphate transfer, etc. The transfer method can be selected to ensure stable expression of the nucleic acid encoded by the fHbp to be introduced. The fHbp-encoding nucleic acid can be provided as a heritable free element (e.g., a plasmid) or integrated into the genome.

[0241] In the composition, the suitable vector for transferring the fHbp-encoded nucleic acid can be modified. The integrative vector can be conditionally replicated, or a suicide plasmid, bacteriophage, etc. The construct can include a variety of elements, including, for example, promoters, selective genetic markers (e.g., genes resistant to antibiotics such as kanamycin, erythromycin, chloramphenicol, or gentamicin), origins of replication (initiating replication in the host cell, e.g., bacterial host cells), etc. The choice of vector depends on various factors, such as the cell type and reproductive purpose for which propagation is desired. Some vectors can be used to amplify and prepare large quantities of the target DNA sequence. Other vectors are suitable for expression in cultured cells. Still others are suitable for transfer and expression in whole animal cells. Selecting a suitable vector is a well-known technique in the art. Many such vectors are commercially available.

[0242] In one example, the vector is an expression vector based on an addendum plasmid containing selectable drug resistance markers and elements and capable of self-replication in different host cells (e.g., Escherichia coli and Neisseria meningitidis). An example of such a “shuttle vector” is the plasmid pFP10 (Pagotto et al. (2000) Gene 244:13-19).

[0243] The construct can be prepared by, for example, inserting the target polynucleotide into the construct backbone, typically by using DNA ligase to ligate it to a restriction enzyme site in the vector. Alternatively, the target nucleotide sequence can be inserted via homologous recombination or site-specific recombination. Homologous recombination is usually accomplished by ligating the homologous region to the vector flanking the target nucleotide sequence, while site-specific recombination is accomplished by using sequences that promote site-specific recombination (e.g., cre-lox, att sites, etc.). The nucleic acid containing this sequence can be added, for example, by ligating an oligonucleotide, or by using a polymerase chain reaction with primers containing both a homologous region and a portion of the target nucleotide sequence.

[0244] Vectors can remain extrachromosomally within host cells or integrate into the host cell genome. Numerous publications well-known to those skilled in the art describe vectors in detail, including, for example, *Short Protocols in Molecular Biology*, (1999) F. Ausubel, et al., eds., Wiley & Sons. Vectors can express nucleic acids encoding target fHbp, amplify said target nucleic acids, or both.

[0245] Exemplary vectors that may be used include, but are not limited to, vectors derived from recombinant phage DNA, plasmid DNA, or coplasmal DNA. For example, plasmid vectors such as the pBR322, pUC 19 / 18, pUC 118, 119, and M13 mp series vectors may be used. pET21 is also an expression vector that may be used. Phage vectors may include the λgt10, λgt11, λgt18-23, λZAP / R, and EMBL series phage vectors. Other 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 charonid 9 series vectors.

[0246] For the expression of the target fHbp, an expression cassette can be used. Therefore, this invention provides a recombinant expression vector containing the target nucleic acid. The expression vector provides transcriptional and translational regulatory sequences and can provide inducible or constitutive expression, wherein operationally linked coding regions are located under transcriptional control in transcriptional initiation and transcriptional termination regions. These control regions may be inherent to the target fHbp from which it is derived, or may be derived from an external source. Typically, the transcriptional and translational regulatory sequences include, but are not limited to, promoter sequences, ribosome binding sites, transcription initiation and termination sequences, translation initiation and termination sequences, and enhancer or activator sequences. The promoter can be constitutive or inducible, and can be a strongly constitutive promoter (e.g., T7, etc.).

[0247] Expression vectors typically have convenient restriction sites located near the promoter sequence for inserting nucleic acid sequences encoding the target protein. Operable selection markers may be present in the expression host to facilitate selection of the cells containing the vector. Furthermore, the expression construct may include other elements. For example, the expression vector may have one or two replication systems, thereby enabling its retention in the organism used for expression, such as mammalian or insect cells, and in a prokaryotic host for cloning and amplification. Additionally, the expression construct may contain selection marker genes to allow selection of the transformed host cells. Selection genes are common knowledge in the art and vary depending on the host cell used.

[0248] It should be noted that the fHbp of the present invention may include other elements, such as detectable tags, for example, radioactive tags, fluorescent tags, biotin tags, and immunodetectable tags (e.g., HA tags, multihistidine tags), etc. Other elements of the fHbp may be provided to facilitate separation by various methods (e.g., affinity capture, etc.) (e.g., biotin tags, immunodetectable tags). The target fHbp may optionally be fixed to a support by covalent or non-covalent linkage.

[0249] The separation and purification of fHbp can be performed according to methods known in the art. For example, fHbp can be separated from genetically modified cell lysates or synthetic reaction mixtures capable of expressing fHbp by immunoaffinity purification, which typically involves: contacting the sample with an anti-fHbp antibody (e.g., an anti-fHbp mAb, such as JAR 5MAb or other suitable JAR MAb known in the art), washing to remove non-specifically bound material, and eluting specifically bound fHbp. The separated fHbp can be further purified by dialysis and other methods commonly used for protein purification. In one example, metal chelate chromatography can be used to separate fHbp.

[0250] host cells

[0251] A variety of suitable host cells can be used to produce fHbp. Generally, according to conventional techniques, the fHbp described in this invention can be expressed in prokaryotic or eukaryotic cells, commonly bacteria, and more commonly *Escherichia coli* or *Neisseria* species (e.g., *Neisseria meningitidis*). Therefore, this invention further provides genetically modified host cells containing nucleic acids encoding the target fHbp. The host cells used for producing (including large-scale production) the target fHbp can be selected from any of a variety of available host cells. Examples of host cells for expression include cells of prokaryotic or eukaryotic unicellular organisms, such as bacteria (e.g., *Escherichia coli* strains), yeasts (e.g., *Saccharomyces cerevisiae*, *Pichia pastoris*, etc.), and may include host cells derived from higher organisms, such as insects, vertebrates, and especially mammals (e.g., *CHO*, *HEK*, etc.). Typically, bacterial host cells and yeasts are particularly preferred in the production of the target fHbp.

[0252] The target fHbp can be prepared in a fully purified or fully isolated form (i.e., fully isolated from other Neisseria or host cell peptides) or a fully isolated form. The target fHbp can be present in the composition in which the peptide is enriched compared to other potentially present components (e.g., other peptides or other host cell components). Purified target fHbp can be provided such that the peptide is present in the composition substantially free of other expressed peptides, for example, less than 90%, typically less than 60%, and more typically less than 50% of the composition consists of other expressed peptides.

[0253] Host cells used to produce vesicles

[0254] When the target fHbp is provided in membrane vesicles (discussed in detail below), the Neisseria host cell is genetically modified to express the target fHbp. In the method disclosed in this invention, any of a variety of Neisseria strains that can be modified to produce the target fHbp can be used, and optionally, strains capable of producing or modified to produce other target antigens, such as PorA.

[0255] Methods and vectors for genetic modification of Neisseria strains and expression of target peptides are well 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, especially strong constitutive promoters, are particularly preferred. Examples of promoters include porA, porB, lbpB, tbpB, p110, hpuAB, lgtF, opa, p110, lst, hpuAB, and rmp promoters.

[0256] For use in membrane vesicle production, pathogenic Neisseria strains or strains derived from the Neisseria genus are preferred, especially strains pathogenic to humans or derived from strains pathogenic to or symbiotic with humans. Examples of Neisseria include *Neisseria meningitidis*, *Neisseria aurea*, *Neisseria gonorrhoeae*, *Neisseria lactis*, *Neisseria polysaccharide*, *Neisseria griseus*, *Neisseria mucosae*, *Neisseria flavescens*, *Neisseria xanthophyllosa*, *Neisseria rhizoma*, *Neisseria longiformis*, etc.

[0257] Genetically modified Neisseria meningitidis strains are particularly preferred for expressing the target fHbp and producing vesicles. Strains for vesicle production can be selected based on a variety of different target characteristics. For example, strains can be selected based on: the type of target PorA (“serotype,” as described above), capsule group, serotype, etc.; reduced production of capsular polysaccharides, etc. For example, the producing strain can produce any target PorA polypeptide and can express one or more PorA polypeptides (natural or genetically engineered). Examples of such strains include strains that produce PorA polypeptides conferring serotypes P1.7,16; P1.19,15; P1.7,1; P1.5,2; P1.22a,14; P1.14; P1.5,10; P1.7,4; P1.12,13; and strains that produce variants of such PorA polypeptides, which may or may not retain reactivity with conventional serological reagents used in serotype typing. Also preferred are PorA peptides identified based on the porA variable region (VR) (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 large number of different VR types have been identified, which can be classified into the VR1 and VR2 family "prototypes". Web-access databases describing this terminology and its relationship to previous typing schemes can be found at neisseria.org / nm / typing / pora. For comparisons of certain VR1 and VR2 types of PorA, see Russell et al. (2004) Emerging Infect Dis 10:674-678.

[0258] Optionally or additionally, the producing strain may be a capsular-deficient strain. Capsular-deficient strains can provide vesicular vaccines that can reduce the risk of triggering significant autoantibody responses in vaccine recipients (e.g., due to the production of antibodies that cross-react with sialic acid on the surface of host cells). As used herein, “capsular-deficient” or “lacking capsular polysaccharide” means that the level of capsular polysaccharide on the bacterial surface is lower than that of naturally occurring strains, or, when the strain is genetically modified, the level of capsular polysaccharide on the bacterial surface is lower than that of parental strains from which the capsular-deficient strain was derived. Capsular-deficient strains include strains with a reduction of at least 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, 90% or more in the production of surface capsular polysaccharides, and also include strains from which capsular polysaccharides are undetectable on the bacterial surface (e.g., by using a whole-cell enzyme-linked immunosorbent assay (ELISA) with anti-capsular polysaccharide antibodies).

[0259] Capsule-deficient strains include strains that lack a capsule due to genetic modifications, either naturally occurring or recombinant. Naturally occurring capsule-deficient strains (see, for example, Dolan-Livengood et al. (2003) J. Infect. Dis. 187:1616-28), and methods for identifying and / or generating 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 well known in the art.

[0260] Modifying Neisseria host cells to reduce capsular polysaccharide production may include modifying one or more genes involved in capsular synthesis, such modification reducing capsular polysaccharide levels compared to the parental cells before modification. This genetic modification includes altering the nucleotide and / or amino acid sequences of one or more capsular biosynthesis genes, causing the strain to lack a capsule (e.g., due to one or more insertions, deletions, substitutions, etc., in one or more capsular biosynthesis genes). Capsular-deficient strains may lack one or more capsular genes, or the genes may be nonfunctional.

[0261] Particularly preferred are strains defective in the biosynthesis of sialic acid. Such strains can produce vesicles that reduce the risk of inducing anti-sialic acid antibodies that cross-react with human sialic acid antigens, and these strains can also improve production safety. Strains with defects in sialic acid biosynthesis (due to naturally occurring modifications or engineered modifications) can be defective in any of the many different genes in the sialic acid biosynthetic pathway. Particularly preferred are strains with defects in the gene product encoded by the N-acetylglucosamine-6-phosphate 2-epimerase gene (referred to as synX AAF40537.1 or siaA AAA20475), and especially preferred are strains with inactivated synX gene. For example, in one embodiment, a capsular-deficient strain is generated by disrupting the production of a functional synX gene product (see, for example, Swartley et al. (1994) J Bacteriol. 176:1530-4).

[0262] Non-recombinant techniques can also be used to obtain capsular-deficient strains from naturally occurring strains, for example, by using bactericidal anti-capsular antibodies to select strains with reduced capsular polysaccharides.

[0263] When the present invention relates to the use of two or more strains (e.g., to produce an antigenic composition comprising vesicles with target fHbp from different strains), strains that differ in one or more strain characteristics may be selected, for example, to provide vesicles with different target fHbp, PorA, etc.

[0264] Vesicle preparation

[0265] The antigenic compositions described in this invention typically comprise vesicles prepared from Neisseria cells expressing the target fHbp. The term "vesicle" as used in this application includes outer membrane vesicles and microvesicles (also known as blebs).

[0266] The antigenic composition may comprise outer membrane vesicles (OMVs) prepared from the outer membrane of a genetically modified strain of *Neisseria meningitidis* expressing the target fHbp. OMVs can be obtained by culturing *Neisseria meningitidis* in broth or solid medium, preferably by separating bacterial cells from the medium (e.g., by precipitating cells through filtration or low-speed centrifugation, etc.), lysing cells (e.g., by adding detergent, osmotic shock, sonication, cavitation, homogenization, etc.), and separating outer membrane components from cytoplasmic molecules (e.g., by filtration; or by differential precipitation or aggregation of the outer membrane and / or outer membrane vesicles; or by using affinity separation methods with ligands that specifically recognize outer membrane molecules; or by high-speed centrifugation to precipitate the outer membrane and / or outer membrane vesicles, etc.); OMVs can be prepared using the outer membrane components.

[0267] The antigenic composition may comprise microvesicles (MVs) (or “vesicles”) containing the target fHbp, wherein a genetically modified Neisseria meningitidis strain expressing the target fHbp releases the MVs or vesicles during culture. For example, MVs can be obtained by culturing Neisseria meningitidis in a broth medium, isolating intact cells from the broth medium (e.g., by filtration or low-speed centrifugation to precipitate only the cells, but not the smaller vesicles, etc.), and then collecting the MVs from a cell-free medium (e.g., by filtration, differential precipitation, or aggregation of MVs, or by high-speed centrifugation to precipitate the vesicles, etc.). The strain used to prepare the MVs is typically selected based on the number of vesicles produced in the medium (e.g., a reasonable number of bacteria can be cultured to prepare vesicles suitable for isolation and administration in the methods described herein). Exemplary strains capable of producing high levels of vesicles are described in PCT Publication No. WO 01 / 34642. In addition to selecting strains for vesicle production based on vesicle yield, strains for MV production can also be selected based on NspA yield, with strains that produce high levels of NspA being particularly preferred (for example, Neisseria meningitidis with varying NspA production levels, see, for instance, Moe et al. (1999 Infect. Immun. 67:5664). Other target strains for vesicle production include strains with an inactive GNA33 gene that encodes a lipoprotein required for cell separation, membrane structure, and virulence (see, for instance, Adu-Bobie et al. (2004) Infect Immun. 72:1914-1919).

[0268] The antigenic compositions of this invention may contain vesicles from one strain, or two, three, four, five, or more strains, which may be homologous or heterologous to each other, typically heterologous. For example, the strains may be homologous or heterologous in relation to PorA and / or the target fHbp from which it is derived. Vesicles may be prepared using strains expressing more than one target fHbp (e.g., one, two, three, or more target fHbps), which may consist of the amino acid sequences of different variants (v.1, v.2, or v.3) or subvariants (e.g., subvariants of v.1, v.2, or v.3) of fHbp.

[0269] The antigenic composition may comprise a mixture of OMVs and MVs providing the same or different target fHbp, wherein the target fHbp may optionally provide epitopes from different combinations of fHbp variants and / or subvariants, and wherein the OMVs and / or MVs may be derived from the same or different strains. Vesicles from different strains may be administered as a mixture or sequentially.

[0270] When necessary (e.g., when the strain used to produce vesicles carries endotoxins or particularly high levels of endotoxins), the vesicles may optionally be treated to reduce endotoxins, for example, to reduce post-administration toxicity. Endotoxins can be reduced by extraction with suitable detergents (e.g., BRIJ-96, sodium deoxycholate, sodium lauroyl sarcosinate, Empigen BB, Triton X-100, Tween 20 (polyoxyethylene sorbitan monolaurate), Tween 80, at concentrations of 0.1-10%, preferably 0.5-2%, and SDS), although this may have limitations as described below. When using detergents for extraction, it is preferable to use detergents other than deoxycholates.

[0271] Vesicles of antigenic compositions can be prepared without detergents, for example, without deoxycholates. While detergent treatment can remove endotoxin activity, the extraction step during vesicle product preparation may remove native fHbp lipoproteins and / or target fHbp (including esterified fHbp). Therefore, detergent-free techniques are particularly needed to reduce endotoxin activity. In one method, strains that produce less endotoxin (lipopolysaccharide, LPS) are used, thereby avoiding the removal of endotoxins from the final product before use in humans. For example, vesicles can be prepared using Neisseria spp. mutants in which lipopolysaccharides or other antigens that may be harmful in vaccines (e.g., Rmp) are reduced or eliminated.

[0272] Vesicles can be prepared using genetically modified Neisseria meningitidis strains to reduce or render undetectable the toxic activity of lipid A. For example, genetic modifications can be made to the biosynthesis of lipid A in these strains (Steeghs et al. (1999) Infect Immun67:4988-93; van der Ley et al. (2001) Infect Immun69:5981-90; Steeghs et al. (2004) J Endotoxin Res10:113-9; Fissha et al. (2005) Infect Immun73:4070). Immunogenic compositions can be detoxified by modifying LPS, for example, by downregulating and / or inactivating enzymes encoding lpxL1 or lpxL2, respectively. The production of pentacylated lipid A in the lpxL1 mutant suggests that the enzyme encoded by lpxL1 adds a C12 to an N-linked 3-OH C14 at the 2' position of GlcN II. In the lpxL2 mutant, the predominant lipid A type is a tetraacylated compound, suggesting that the enzyme encoded by lpxL2 has an additional C12 at the 2-position of GlcN I, i.e., an addition to an N-linked 3-OH C14. Mutations lead to decreased (or absent) expression of these genes (or reduced or inactive products of these genes) and alter the toxicity of lipid A (van der Ley et al. (2001) Infect Immun69:5981-90). Tetraacylated (lpxL2 mutant) and pentaacylated (lpxL1 mutant) lipid A are less toxic than wild-type lipid A. Mutations in the lipid A 4'-kinase encoding gene (lpxK) also reduce the toxic activity of lipid A. For vesicle (e.g., MV or OMV) production, genetically modified Neisseria meningitidis are particularly preferred, resulting in a reduction or undetectability of the protein encoded by the functional LpxL1 gene. For example, genetic modification of Neisseria species (e.g., Neisseria meningitidis strains) can reduce or eliminate the activity of the lpxL1 gene product. For instance, Neisseria species can be genetically modified to knock out the lpxL1 gene, for example, by disrupting the lpxL1 gene. See, for example, U.S. Patent Publication No. 2009 / 0035328. Neisseria species can be genetically modified to reduce or eliminate the activity of the lpxL2 gene product. Neisseria species can also be genetically modified to reduce or eliminate the activity of both the lpxL1 and lpxL2 gene products. Compared to wild-type Neisseria meningitidis strains capable of producing LPS, these vesicles exhibit reduced toxicity but retain the immunogenicity of fHbp.

[0273] The toxicity of LPS can also be altered by introducing mutations into genes / locuses involved in polymyxin B resistance (which is associated with the addition of aminoarabinose to the 4' phosphate group of lipid A). These genes / locuses can be pmrE encoding UDP-glucose dehydrogenase, or a region of a common antimicrobial peptide resistance gene shared by many enterocins involved in the synthesis and transfer of aminoarabinose. The pmrF gene, present in this region, encodes a polyterpene phosphate mannosyltransferase (Gunn JS, Kheng, BL, Krueger J., Kim K., Guo L., Hackett M., Miller S.I. 1998. Mol. Microbiol. 27:1171-1182).

[0274] Mutations in the PhoP-PhoQ regulatory system, which is a phosphorylated two-component regulatory system (e.g., the PhoP constitutive phenotype, PhoPc), or low Mg++ environments or culture conditions (which activate the PhoP-PhoQ regulatory system), can lead to the addition of aminoarabinose to the 4' phosphate and the substitution of nutmeg with 2-hydroxymyristate (nutmeg hydroxylation). This modified lipid A has a reduced ability to stimulate human endothelial cells to express E-selectin and to stimulate human monocytes to secrete TNF.

[0275] Polymyxin B-resistant strains are also applicable because these strains exhibit reduced LPS toxicity (see, for example, van der Ley et al. (1994) In: Proceedings of the ninth international pathogenic Neisseria conference. The Guildhall, Winchester, England). Alternatively, a synthetic peptide that mimics polymyxin B binding activity may be added to the antigenic composition to reduce LPS toxicity (see, for example, Rustici et al. (1993) Science 259:361-365; Porro et al. (1998) Prog Clin Biol Res. 397:315-25).

[0276] Endotoxins can also be reduced by selecting appropriate culture conditions. For example, culturing the strain in a growth medium containing 0.1 mg–100 mg of aminoarabinose per liter can reduce lipid toxicity (see, for example, WO 02 / 097646).

[0277] Compositions and Formulations

[0278] As used herein, the terms "composition," "antigen composition," "antigenic composition," or "immunogenic composition" generally refer to compositions comprising the target fHbp described in this invention, which may optionally be conjugated to further enhance immunogenicity. This invention particularly includes compositions for inducing antibodies in humans, such as antibodies against Neisseria meningitidis, or bactericidal antibodies against Neisseria meningitidis. Antigenic compositions may comprise one, two, or more different target fHbps. When more than one type of fHbp is present, each target fHbp may provide epitopes from different combinations of fHbp variants and / or subvariants.

[0279] The antigenic composition contains an immunologically effective amount of the target fHbp, and may further contain other compatible components if necessary. The compositions of the present invention may contain an fHbp as a low-binding fH protein. The low-binding fH protein in the target composition includes any fHbp described above, such as non-naturally occurring or naturally occurring fHbp (e.g., fHbp ID 14 and / or fHbp ID 15). The composition contains one or more fHbps, wherein at least one fHbp is a low-binding fH protein. When the composition contains more than one fHbp, the fHbps may be different (e.g., different amino acid sequences and / or conjugates).

[0280] The immunogenic compositions of the present invention include, but are not limited to, compositions comprising the following:

[0281] 1) Non-naturally occurring fHbp (e.g., non-naturally occurring fHbp with a lower affinity for human fH than fHbp ID 1); and

[0282] 2) fHbp (e.g., non-naturally occurring fHbp, such as non-naturally occurring fHbp with an affinity for fH lower than fHbp ID 1) and NspA;

[0283] The fHbp and / or NspA may be provided as recombinant proteins and / or in vesicle compositions (e.g., OMV). It should be noted that when the composition includes NspA and fHbp, the bactericidal activity of the antibody induced by administration of the composition is the result of the combined action of antibodies against one or both antigens. Examples of immunogenic compositions provided by the present invention include:

[0284] a) Immunogenic compositions comprising the non-naturally occurring fHbp variants described above (e.g., non-naturally occurring fHbp that elicits a bactericidal antibody response against at least one strain of Neisseria meningitidis);

[0285] b) Immunogenic compositions comprising the above-described non-naturally occurring fHbp variants (e.g., non-naturally occurring fHbp with a lower affinity for human fH than fHbp ID 1); and recombinant NspA protein;

[0286] c) An immunogenic composition comprising isolated fHbp, wherein the isolated fHbp has at least 85% amino acid sequence identity with fHbp ID 14 or fHbp ID 15, wherein the fHbp has a lower affinity for human factor H (fH) than fHbp ID 1.

[0287] d) An immunogenic composition comprising isolated fHbp and recombinant NspA protein; wherein the isolated fHbp has at least 85% amino acid sequence identity with fHbp ID 14 or fHbp ID 15, wherein the fHbp has a lower affinity for human factor H (fH) than fHbp ID 1;

[0288] e) An immunogenic composition comprising a natural OMV derived from a genetically modified Neisseria host cell, said host cell being genetically modified with nucleic acids encoding a non-naturally occurring fHbp variant as described above (e.g., a non-naturally occurring fHbp with an affinity for human fH lower than fHbp ID 1), such that the genetically modified host cell produces an encoded non-naturally occurring fHbp, wherein said OMV comprises the encoded non-naturally occurring fHbp; and

[0289] f) An immunogenic composition comprising a natural OMV derived from genetically modified Neisseria host cells, said host cells being genetically modified with nucleic acids encoding a non-naturally occurring fHbp variant as described above (e.g., a non-naturally occurring fHbp with a lower affinity for human fH than fHbp ID 1), such that the genetically modified host cells produce an encoding of a non-naturally occurring fHbp, wherein said OMV contains the encoding of a non-naturally occurring fHbp; and said Neisseria host cells also produce high levels of NspA, such that said OMV also contains NspA. For example, said Neisseria host cells may be genetically modified to increase NspA expression.

[0290] "Immuneally effective dose" means that when this dose is administered to an individual, whether as a single dose or as part of a series of administrations of the same or different immunogenic compositions, it is effective in evoking an effective antibody response for the treatment or prevention of symptoms or disease caused by, for example, an infection of the spp. genus Neisseria, particularly Neisseria meningitidis, and more particularly Neisseria meningitidis group B. This dose varies depending on factors such as the health and physical condition of the individual being treated, age, the individual's immune system's ability to produce antibodies, the level of protection targeted, the vaccine formulation, the attending physician's assessment of the medical condition, and other relevant factors. The dose is expected to be within a relatively wide range, which can be determined by routine testing.

[0291] The amino acid sequence of the NspA polypeptide is well 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. The term "NspA polypeptide" may comprise an amino acid sequence that is consistent with... Figure 44 The continuous chain of about 75 amino acids to about 100 amino acids, about 100 amino acids to about 150 amino acids, or about 150 amino acids to about 174 amino acids in the amino acid sequence shown has 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. The “NspA polypeptide” may contain an amino acid sequence that is similar to... Figure 44 The continuous chain of about 75 to about 100 amino acids, or about 100 to about 155 amino acids, of the 20 to 174 amino acids shown in the amino acid sequence has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity. In some cases, the NspA polypeptide lacks a signal sequence; in other cases (e.g., expressed in host cells), the NspA polypeptide includes a signal sequence.

[0292] Dosing regimens can be single-dose or multi-dose regimens (e.g., including booster doses), wherein the multi-dose regimen administers a unit dosage form of the antigenic composition at different times. As used herein, the term "unit dosage form" refers to a physically discontinuous unit suitable for human and animal subjects, each unit containing a predetermined amount of the antigenic composition of the invention sufficient to produce the target effect, provided in combination with pharmaceutically acceptable excipients (e.g., pharmaceutically acceptable diluents, carriers, or solvents). The antigenic composition may be administered in combination with other immunomodulators.

[0293] The antigenic composition is provided in a pharmaceutically acceptable excipient or powder, said excipient being a solution, such as a sterile aqueous solution, typically a physiological saline solution. If necessary, said excipient is substantially inert.

[0294] In some embodiments, the object immunogenic composition comprises object fHbp present in vesicles. In some embodiments, the object immunogenic composition comprises object fHbp present in MV. In some embodiments, the object immunogenic composition comprises object fHbp present in OMV. In some embodiments, the object immunogenic composition comprises a mixture of MV and OMV containing object fHbp. Vesicles, such as MV and OMV, have been described above.

[0295] The antigenic composition may further contain an adjuvant. Examples of known suitable adjuvants for human use include, but are not limited to: alum, aluminum phosphate, aluminum hydroxide, MF59 (4.3% w / v squalene, 0.5% w / v Tween 80). TM , 0.5% w / v Span 85), CpG-containing nucleic acids (in which cytosine is unmethylated), QS21, MPL, 3DMPL, Aquilla extract, ISCOMS, LT / CT mutant, poly(D,L-lactide-co-glycolic acid) (PLG) microparticles, Quil A, interleukins, etc. For laboratory animals, Freund's adjuvant, N-acetyl-murayl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-n-murayl-L-alanyl-D-isoglutamine (CGP 11637, referred to as n-MDP), N-acetyl-murayl-L-alanyl-D-isoglutamine-L-alanine-2-(1'-2'-di-palmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP19835A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria in a 2% squalene / Tween 80 emulsion: monophosphoryl lipid A, trehalose dimethicone ester, and cell wall skeleton (MPL+TDM+CWS), can be used. The potency of the adjuvant can be determined by detecting the amount of antibody against the immunogenic antigen or its epitope.

[0296] Further exemplary adjuvants that can enhance the potency of the composition include, but are not limited to: (1) oil-in-water emulsions (containing or excluding other specific immunostimulants, such as muramyl peptides (see below) or bacterial cell wall components), such as example (a) MF59 TM(a) 5% squalene, 0.5% Tween 80 and 0.5% Span 85 (optionally containing MTP-PE) (WO 90 / 14837; Chapter 10 in Vaccine Design: The Subunit and Adjuvant Approach, eds. Powell & Newman, Plenum Press 1995), prepared into submicron particles using a microfluidic apparatus; (b) SAF, containing 10% squalene, 0.4% Tween 80, 5% Prönnicke block polymer L121, and thr-MDP, microfluidized into a submicron emulsion, or vibrated to produce an emulsion with a larger particle size; and (c) RIBI TM The adjuvant system (RAS) (Ribi Immunochem, Hamilton, Mont.) contains 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components, such as monophosphatidyllipid A (MPL), trehalose dimethicone ester (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox). TM (2) Saponin adjuvants, such as QS21 or Stimulon, can be used. TM(Cambridge Bioscience, Worcester, Mass.) or particles derived therefrom, such as ISCOM (immunostimulatory complexes), which may be lacking additional detergents, such as WO 00 / 07621; (3) complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA); (4) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 (WO99 / 44636) etc.), interferons (e.g., gamma interferon), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; (5) monophospholipid A (MPL) or 3-O-deacylated MPL (3dMPL), such as see GB-2220221, EP-A-0689454, optionally, when used in combination with pneumococcal sugar, substantially free of alum, such as WO 00 / 56358; (6) Compositions of 3dMPL with, for example, QS21 and / or oil-in-water emulsions, such as EP-A-0835318, EP-A-0735898, EP-A-0761231; (7) Oligonucleotides containing CpG motifs (Krieg Vaccine 2000, 19, 618-622; Krieg Curr opin Mol Ther 2001 3:15-24; Roman et al., Nat. Med, 1997, 3, 849-854; Weiner et al., PNAS USA, 1997, 94, 10833-10837; Davis et al., J. Immunol, 1998, 160, 810-876; Chu et al., J. Exp. Med, 1997, 186,1623-1631;Lipford et al,Ear.J.Immunol.,1997,27,2340-2344;Moldoveami e / al.,Vaccine,1988,16,1216-1224,Krieg et al.,Nature,1995,374,546-549;Klinman et al. al., PNAS USA, 1996, 93, 2879-2883; Ballas et al, J. Immunol, 1996, 157, 1840-1845; Cowdery et al, J. Immunol, 1996, 156, 4570-4575; Halpern et al, Cell Immunol,1996,167,72-78; Yamamoto et al,Jpn.J.Cancer Res.,1988,79,866-873;Stacey et al,J.Immunol.,1996,157,2116-2122;Messina et al,J.Immunol,1991,147,1759-1764;Yi et al,J.Immunol,1996,157,4918-4925;Yi et al, J. Immunol, 1996, 157, 5394-5402; Yi et al, J. Immunol, 1998, 160, 4755-4761; and Yi et al, J. Immunol, 1998, 160, 5898-5906; international patent applications WO 96 / 02555, WO 98 / 16247, WO 98 / 18810、WO 98 / 40100, WO 98 / 55495, WO 98 / 37919 and WO 98 / 52581), i.e., containing at least one CG dinucleotide in which cytosine is unmethylated; (8) polyoxyethylene ethers or polyoxyethylene esters, such as WO 99 / 52549; (9) compositions of polyoxyethylene sorbitan ester surfactants with octyl phenyl alcohol (WO 01 / 21207), or compositions of polyoxyethylene alkyl ethers or ester surfactants with at least one other nonionic surfactant, such as octyl phenyl alcohol (WO 01 / 21152); (10) saponins and immunostimulatory oligonucleotides (e.g., CpG oligonucleotides) (WO 00 / 62800); (11) immunostimulants and metal salt particles, such as WO 00 / 23105; (12) saponins and oil-in-water emulsions, such as WO 99 / 11241; (13) saponins (e.g., QS21) + 3dMPL + IM2 (optional + hormone), e.g., WO 98 / 57659; (14) other substances used as immunostimulants to enhance the potency of the composition. Muraminoyl peptides include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-25-acetyl-normumamyl-L-alanyl-D-isoglutamine (norm-MDP), N-acetyl-muramyl-L-alanyl-D-isoglutamine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), etc. Adjuvants suitable for human administration are particularly preferred.

[0297] The antigen composition may contain other components, such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium, carbonates, etc. The composition may contain pharmaceutically acceptable excipients to mimic physiological conditions, such as pH adjusters and buffers, toxicity modifiers, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.

[0298] The concentration of the target fHbp in the formulation can vary widely (e.g., on a weight basis, from less than about 0.1%, typically or at least about 2% to up to 20% to 50% or higher), primarily depending on the specific route of administration chosen and the patient’s needs, based on fluid volume, viscosity, and patient-based factors.

[0299] Formulations containing fHbp can be provided in the form of solutions, suspensions, tablets, pills, capsules, powders, gels, creams, lotions, ointments, aerosols, etc. It is known that when administered orally, the composition needs to be protected from digestion. This is generally achieved by formulating the composition resistant to acid and enzymatic degradation, or by coating the composition with a suitable resistant carrier. Methods for protecting against digestion are common knowledge in the art.

[0300] Formulations containing fHbp may also be provided, such that the serum half-life of fHbp is increased after administration. For example, when fHbp alone is formulated as an injection, it can be formulated as a liposomal formulation, a colloid, or other conventional formulation capable of prolonging the serum half-life. Liposomes can be prepared using various methods; see, for example, Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), US Patent Nos. 4,235,871, 4,501,728, and 4,837,028. The formulations may also be formulated as controlled-release or sustained-release formulations.

[0301] Immunization

[0302] This invention provides a method for inducing an immune response against at least one strain of Neisseria in a mammalian host. The method typically involves administering an effective amount of a target immunogenic composition to the desired individual.

[0303] Generally, an antigenic composition containing fHbp is administered to individuals at risk of acquiring Neisseria gonorrhoeae disease to prevent or at least partially halt the development of the disease and its complications. The dosage sufficient to achieve this purpose is defined as the "therapeutic effective dose." Therapeutic effective doses depend on factors such as the antigenic composition, the route of administration, the patient's weight and overall health condition, and the prescribing physician's judgment. Whether the antigenic composition is administered as a single dose or multiple doses depends on the patient's required and tolerable dose and frequency, as well as the route of administration.

[0304] Typically, an effective amount of an antigenic composition containing fHbp is administered to the host to elicit an immune response, particularly a humoral immune response, such as a bactericidal antibody response. As mentioned above, the dosage of immunization can vary, typically ranging from about 1 μg to 100 μg / 70 kg patient, more commonly 5 μg to 50 μg / 70 kg. Oral, nasal, or topical administration generally allows for higher doses (e.g., 10 mg to 100 mg or higher). Booster immunizations can be given after the initial dose with the same or different antigenic compositions containing fHbp. Usually, at least one booster dose is given, more commonly two.

[0305] Generally, immunization can be administered via any suitable route, including oral, nasal, nasopharyngeal, parenteral, intestinal, gastric, local, subcutaneous, and intramuscular administration, in tablet, solid, powder, liquid, or aerosol form, for local or systemic administration, with or without excipients. For a more detailed description of practical methods for preparing parenteral-administerable compositions known or understood by those skilled in the art, see, for example, Remington's Pharmaceutical Science, 15th edition, Mack Publishing Company, Easton, Pa. (1980).

[0306] The immune response to anti-fHbp can be assessed using known methods (e.g., by collecting serum from individuals before and after the first immunization and demonstrating changes in the individual's immune status using methods such as immunoprecipitation, ELISA, bactericidal assay, Western blot, or flow cytometry).

[0307] The antigenic composition can be administered to human subjects who are not immunologically naive. In one particular embodiment, the subject is a child about 5 years of age or younger, preferably about 2 years of age or younger, and the antigenic composition is administered at one or more of the following times: two weeks after birth, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months, or 1 year, 15, 18, or 21 months, or at 2, 3, 4, or 5 years of age.

[0308] It is generally desirable to administer the first immunization before the first signs of disease symptoms appear, or when the first signs of possible or actual exposure to infection or disease (e.g., due to exposure to or infection with Neisseria) appear.

[0309] Filtering methods

[0310] In one example, a method for assessing the effectiveness of the target fHbp in a vaccine composition includes: (a) immunizing a host animal (e.g., a non-human mammalian host animal, such as a rodent, such as a mouse) with a composition containing the fHbp of the present invention; and (b) detecting the level of bactericidal antibodies in the host. The target method may also include assessing the host animal's susceptibility to bacterial pathogens by administering a vaccine containing the target fHbp to the host animal.

[0311] In another example, the method may include preparing and identifying antibodies triggered by the target fHbp. The method includes isolating an antibody with binding affinity for fHbp from a host animal, contacting bacterial cells with the isolated antibody, and evaluating the binding of the antibody to the bacterial cells. Additional steps may include evaluating the competitive binding of the antibody against fHbp to human factor H; and evaluating bactericidal activity against the bacterial pathogen when the antibody is administered to an animal with a bacterial pathogen. In some embodiments, the antibody is in a population of antibodies, and the method may further include isolating one or more antibodies that bind to the bacterial cells from the population of antibodies. A distinctive aspect is that the isolated antibody has bactericidal activity against bacterial cells, for example, through complement-mediated bactericidal activity and / or phage activity, which can reduce the viability of bacteria in human blood.

[0312] The preferred bacterial pathogens are any or all variants of various capsular groups of Neisseria meningitidis, such as Neisseria meningitidis serogroup B, Neisseria meningitidis serogroup C, Neisseria meningitidis serogroup X, Neisseria meningitidis serogroup Y, Neisseria meningitidis serogroup W-135, etc.

[0313] Assessment F H BP Response methods

[0314] The present invention provides a method for determining the likelihood of fHbp eliciting a bactericidal response in an individual; and a method for evaluating fHbp variants suitable for inclusion in immunogenic compositions.

[0315] Determine the likelihood that fHbp will induce a bactericidal response.

[0316] This invention provides a method for determining the likelihood that fHbp (e.g., fHbp present in Neisseria spp. vaccines) will elicit a bactericidal response against at least one strain of Neisseria meningitidis in an individual. The method generally includes determining the ability of an antibody to inhibit the binding of fH to fHbp, said antibody being present in serum obtained from an individual immunized with fHbp. The level of inhibition of fH to fHbp binding by said antibody is at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 2 times, at least about 10 times, at least about 50 times, at least about 100 times, or greater than 100 times that of a control antibody, where the control antibody inhibits the binding of fH to fHbp but does not elicit a bactericidal response, indicating that fHbp may elicit a bactericidal response against at least one strain of Neisseria meningitidis. In some embodiments, as described above, the fHbp is a non-naturally occurring fHbp that has a lower affinity for human H factor (fH) than fHbp ID 1.

[0317] The extent to which the antibody-induced inhibition of fH / fHbp binding by the fHbp variant can be determined using the detection method described in this application or any other known detection method. For example, the fH and / or the fHbp may contain a detectable label, and the inhibition of fH / fHbp binding can be determined by detecting the amount of the labeled component present in the fH / fHbp complex, and / or by detecting the amount of the label present in free fH and / or free fHbp (e.g., fH or fHbp not present in the fH / fHbp complex).

[0318] In one example, a method for assessing the binding of fH to fHbp includes using fHbp immobilized on a support (e.g., fHbp immobilized in the wells of an ELISA plate). A mixture containing a fixed concentration of human fH and a dilution of the antibody to be tested (e.g., antiserum, such as from a human or non-human test animal (e.g., a mouse) that has received an immunogenic composition after an antibody challenge dose) is added to the wells and incubated for a sufficient time to allow antibody binding. After washing the wells, the bound fH is detected using a specific anti-fH antiserum (e.g., goat or donkey) containing a labeled component, or a second labeled antibody (e.g., rabbit anti-goat or anti-donkey antiserum). The percentage of fH binding inhibition can be calculated by the amount of fH bound without the addition of human or mouse antibodies.

[0319] In another variation of this type of assay, flow cytometry is used to assess the binding of fH to live bacteria in the presence or absence of the test antiserum. Bacterial cells are co-incubated with different dilutions of a fixed concentration of fH (e.g., a detectable labeled fH) and the test serum containing the antibody. The bacteria are washed and the bound fH is detected (e.g., as described above).

[0320] Therefore, assessing the ability of antiserum from individuals immunized with fHbp to inhibit fH / fHbp binding can replace directly assessing the bactericidal activity of the antiserum. The method of this invention for determining the likelihood of fHbp eliciting a bactericidal response in an individual can provide information to clinicians or other medical personnel to determine whether a particular immunogenic composition has elicited an effective bactericidal response in an individual.

[0321] Immunized individuals with similar serum IgG anti-fHbp antibody titers were measured using ELISA. If the antiserum provided better overall fH binding inhibition, it indicated a more effective, higher-quality anti-fHbp antibody response, providing better protection. Therefore, for example, when comparing anti-Neisseria spp. antibody responses in two individuals (by comparing anti-fHbp antibodies, i.e., comparing inhibition of a 1:10,000 serum dilution with that of another individual at a 1:3,000 dilution), the individual with higher inhibitory activity had higher-quality anti-fHbp antibodies and would provide better protection. Therefore, fH inhibition assays can be used as an alternative to complement-mediated bactericidal titers, which are typically more time-consuming and difficult to detect compared to fH inhibition.

[0322] Evaluation of fHbp variants

[0323] This invention provides a method for assessing or predicting the likelihood of an fHbp variant effectively eliciting a bactericidal antibody response in an individual. The method typically includes assessing the ability of an antibody specific to the fHbp variant to inhibit the binding of fH to fHbp. The inhibitory strength of the antibody evoked by the fHbp variant on the binding of fH to fHbp is positively correlated with the bactericidal activity of the antibody evoked by the fHbp variant. If an antibody evoked by the fHbp variant can inhibit the binding of fH to fHbp at higher serum dilutions, it may be considered a suitable vaccine candidate for inducing protection against one or more Neisseria strains.

[0324] For example, the present invention provides a method for determining the likelihood that a non-naturally occurring fHbp will induce a bactericidal antibody against at least one strain of Neisseria meningitidis in an individual, wherein the fHbp has a lower affinity for human fH than fHbp ID 1. The method typically includes determining the ability of an antibody induced by the non-naturally occurring fHbp in a non-human test animal to inhibit the binding of fH to fHbp. An inhibition level of the antibody induced by the non-naturally occurring fHbp against the binding of fH to fHbp that is at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 2 times, at least about 10 times, at least about 50 times, at least about 100 times, or greater than 100 times that of an antibody induced by fHbp ID 1 in a non-human test animal indicates that the non-naturally occurring fHbp is likely to induce a bactericidal response against at least one strain of Neisseria meningitidis.

[0325] Suitable non-human test animals include, for example, mice, rats, rabbits, etc. The extent to which the antibody induced by the fHbp variant inhibits the binding of fH to fHbp can be determined using the detection method described in this application or any other known detection method. The bactericidal activity of the antibody can be readily determined using the detection method described in this application or any other known detection method.

[0326] A method for determining the likelihood that a given, non-naturally occurring fHbp with an affinity for human fH less than fHbp ID 1 will induce bactericidal antibodies against at least one strain of Neisseria meningitidis in an individual can be used, for example, in vaccine development to identify suitable immunogens (and / or eliminate unsuitable immunogens). Example

[0327] It should be understood that the examples and embodiments described in this application are for illustrative purposes only, and various modifications or variations may be made by those skilled in the art in light of these examples and embodiments. Such modifications and variations should be included within the scope of the concept and rights of this application and the appended claims. All publications, patents, and patent applications referenced in this application are incorporated herein by reference in their entirety for all purposes.

[0328] Implementation Examples Overview

[0329] Factor H (fH) is present in serum at high concentrations (~200 to 800 μg / ml). The binding of fH to fHbp is specific for human fH (Granoff et al. (2009) Infect Immun 77:764). This implies that when humans are immunized with fHbp, the molecule can form a complex with fH. Conversely, when non-human primates or other laboratory animals are immunized, the antigen is present in the immune system but does not bind to fH. In humans, the fH-fHbp complex may affect the immunogenicity of fHbp (e.g., by covering epitopes and influencing antigen presentation).

[0330] This application provides evidence that the presence of human fH reduces the protective antibody response of fHbp vaccines that bind to fH. Furthermore, while some mutant vaccines with one or two amino acid substitutions do not bind to fH (e.g., E218A and / or E239A), the specific mutations used to alter the molecule result in a change that reduces the vaccine's ability to elicit serum bactericidal antibodies. It has been determined that other single amino acid mutants (e.g., R41S or R41A mutants of fHbp ID 1; R41S mutants of fHbp ID 4, 9, 74 or chimeric fHbp I; R130A mutant of fHbp ID 1; R80A, D211A, E218A, E248A, or G236I mutants of fHbp ID 22; T220A / H222A mutant of fHbp ID 22; R41S / K113A, R41S / K119A, R41S / D121A, or R41S / K113A / D121A mutants of fHbp ID 77; and K199A or E218A mutants of fHbp ID 28) bind to fH in a reduced manner. The ability of fHbp vaccines with the R41S mutation to elicit bactericidal antibodies is not reduced, and in the presence of human fH, they exhibit a higher protective antibody response than wild-type fHbp ID1 vaccines that bind to fH. Other mutations, such as K241E in fHbp ID1 or E241K from the crystal structure of fHbp ID15, are expected to engage with fH but have no effect on fH binding. Furthermore, the R41S mutation reduces fH binding in fHbp ID1, 4, 9, and 74, but does not reduce fH binding in fHbp ID22 or 77. Mutations that reduce fH binding but have minimal or no effect on the conformation of fHbp (such as R41S in fHbp ID1, and other mutations discussed below) make mutant vaccines capable of eliciting bactericidal antibodies and can serve as very good vaccine candidates. Therefore, the fHbp variant maintains and presents a conformational epitope that binds to a bactericidal antibody having bactericidal activity against one or more Neisseria meningitidis strains.

[0331] The details of the research of this invention are as follows.

[0332] Materials and methods

[0333] Human fH transgenic mouse model. A 3.9 kbp human complement fH (cfh) cDNA was cloned into plasmid pCAGGS (Niwa et al. (1991) Gene108:193-9). A ~6 kbp SalI-PstI restriction fragment was microinjected into BALB / c mouse embryos and implanted into pseudopregnant female BALB / c mice. The expression of human fH in fetal serum was detected by Western blotting.

[0334] The concentration of human fH in serum. To distinguish human from mouse fH, an enzyme-linked immunosorbent assay (ELISA) specifically binding to human fH was used to capture fHbp. Recombinant fHbp (2 μg / ml) in sterile phosphate-buffered saline (PBS) was added to each well of the ELISA plate. After blocking with 1% bovine serum albumin (BSA), a dilution of unimmunized mouse or human serum was added. Bound human fH was detected using sheep anti-human fH antiserum (Lifespan Biosciences, Seattle, WA; 1:2000 dilution). The ELISA was colorimetrically analyzed using anti-sheep IgG conjugated to alkaline phosphatase. The phosphatase substrate p-nitrophenyl phosphate (Sigma-Aldrich, St. Louis, MO) was added, followed by incubation at room temperature for 30 min, and the absorbance was measured at 405 nm. The concentration of fH was determined by comparison with a human control serum dilution containing 471 μg / ml fH. As a control, fH was detected in 25 serum samples from adult subjects in the San Francisco Bay Area who participated in an IRB-approved trial to screen for normal serum as a complement source for serum bactericidal assays.

[0335] Recombinant fHbp vaccine. Wild-type fHbp and the recombinant R41S mutant protein were purified according to the described method (Beernink PT et al. (2008) Infect Immun76:2568-2575). Immunogenicity of the vaccine was evaluated in 6- to 8-week-old BALB / c wild-type or human fH transgenic mice using a protocol approved by the Animal Feeding and Use Committee of the University of Massachusetts Medical School. Three doses of the vaccine containing 20 μg fHbp, adsorbed onto 600 μg of aluminum hydroxide, were administered intraperitoneally at three-week intervals. The control meningococcal group C conjugate vaccine (Meningitec; Wyeth, Montreal, Canada) contained 2 μg of polysaccharide and 3 μg of CRM adsorbed onto 100 μg of aluminum phosphate. 197 .

[0336] Statistical analysis was performed. A two-tailed Student's t-test was used to compare the reciprocal of the geometric mean titer (GMT) of serum antibody responses between two independent mouse groups. A one-tailed t-test was also used to test whether the anti-fHbp antibody response in transgenic mice immunized with wild-type fHbp vaccine was not lower than that in immunized wild-type mice. A general linear regression model was used to test whether the type of fHbp vaccine and the concentration of human fH affected the serum bactericidal antibody response. To satisfy the normality assumption, the serum bactericidal antibody detection results and fH concentration were logarithmically adjusted in the regression and correlation analyses. 10 Transformation. A two-sided p-value less than or equal to 0.05 was considered statistically significant.

[0337] Example 1: Human F H binding decreases F H BP Immunogenicity of vaccines.

[0338] The binding of fH to fHbp may cover epitopes and weaken antibody responses against the exposed portion of the fHbp molecule on the bacterial surface, which is most effective for bactericidal activity. Because the binding of human fH to fHbp is specific to human fH, the effect of fH on vaccine immunogenicity was studied using a human fH transgenic animal model. The concentration of human fH in the serum of transgenic mice was detected using the fHbp capture ELISA described above, which is specific for human fH. Control wells contained purified human fH at concentrations ranging from 0.15 to 5 μg / ml. Figure 1 (See Figure A). The experimental wells contained different serum dilutions from transgenic mice (serially diluted 2-fold starting at 1:100). In the serum of transgenic mice, the concentration of human factor H was >100 μg / ml. The concentration in serum from H-negative littermates was <12 μg / ml, below the detection limit. Human factor H in wild-type mice is also known to be <12 μg / ml. In contrast, the concentration of fH in stored serum samples from adult complement donors was >100 μg / ml. Figure 1(See Figure B). In the experiments described below, littermates of transgenic mice with <12 μg / ml or known wild-type mice will be referred to as “wild-type” mice.

[0339] In Study 1, transgenic or wild-type human fH mice were immunized with a recombinant fHbp vaccine that binds to human fH (Table 1 below). Three weeks after the third vaccination, the serum bactericidal antibody response in transgenic mice was 8-fold lower than that in wild-type mice where serum fH did not bind to the vaccine (reciprocal of GMT 59 vs. 453 in wild-type mice, P = 0.03). A control vaccine that does not bind to fH was not included in Study 1. Therefore, it should be determined whether the reduced immunogenicity of the fHbp vaccine in transgenic mice is a result of the vaccine antigen binding to human fH, or whether the mice may have a lower serum antibody response to general vaccine antigens. In Study 2, repeated vaccination with fHbp was performed, including the use of a control meningococcal group C polysaccharide-CRM. 197 Transgenic and wild-type mouse groups immunized with a conjugate vaccine, which does not bind to human fH. Representative serum IgG and bactericidal antibody responses in transgenic mice immunized with the meningococcal conjugate vaccine were not significantly different from those obtained in wild-type mice. Figure 2 As observed in Study 1, transgenic mice immunized with the human fH-binding fHbp vaccine in Study 2 had a lower serum bactericidal antibody response (reciprocal of GMT 31 vs. 115 in wild-type mice, P = 0.05, one-sided t-test). Furthermore, there was a negative correlation between the concentration of human fH in the serum of transgenic mice and the serum bactericidal antibody response to the human fH-binding fHbp vaccine. Figure 3 (See Figure A; Pearson correlation coefficient, r = -0.65; P = 0.02). Therefore, the higher the serum concentration of human fH, the lower the serum bactericidal response to the vaccine.

[0340] In both studies, the serum IgG anti-fHbp antibody response in transgenic mice was lower than that in wild-type mice (Study 1, reciprocal of GMT 30,000 vs. 97,000, P = 0.03; Study 2, reciprocal of GMT 107,000 vs. 190,000, P = 0.025). Overall, the data indicate that the conjugation of human fH to fHbp vaccines simultaneously weakens both the titer of IgG anti-fHbp antibodies and the bactericidal antibody response.

[0341] Table 1. Complement-mediated serum bactericidal antibody response in wild-type mice or human fH transgenic mice immunized with a recombinant fHbp vaccine binding to human fH.

[0342]

[0343] WT fHbp vaccine that binds to fH.a,b P = 0.03 (two-sided); c,d P = 0.05 (one-sided), based on the results of Study 1.

[0344] Example 2: Mutations at positions 218 and / or 239 F H BP Leading to F The binding of H is reduced.

[0345] A fHbp mutant with two alanine substitutions at glutamic acid residues 219 and 239 (E218 and E239) was found to eliminate fH binding (Schneider MC et al. (2009) Nature 458:890-3). This was achieved through the previously described Ni... 2+ Recombinant fHbp E218A, E239A, and E218A / E239A mutants were prepared by affinity chromatography (Beernink et al. (2010) Clin Vaccine Immunol 17:1074-8). As described above, the purified recombinant mutants or WT fHbp were used as antigens to measure the binding of human fH to the fHbp mutants by ELISA on an enzyme-labeled plate. This method confirmed that the double mutants reduced the binding of fH (…). Figure 4 Furthermore, the fHbp mutant with a single mutation at E218 or E239 can also reduce the binding of human fH (Figure 4).

[0346] Example 3: F H BP The E218A and / or E239A mutants have weakened immunogenicity in wild-type mice.

[0347] Wild-type ID 1fHbp and E218A / E239A double mutant fHbp were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Ni 2+ Recombinant fHbp expressed in *E. coli* was purified by affinity chromatography, with 5 μg of purified protein loaded onto a gel. NuPAGE 4-12% polyacrylamide gradient gel (Invitrogen, Carlsbad, CA) was then electrophoresed at 200V for 45 min in MES buffer (Invitrogen), followed by staining with Simply Blue Safe Stain (Invitrogen). Molecular weight standards were obtained using Kaleidoscope Broad Range (Bio-Rad, Richmond, CA). Visual inspection of the proteins using Coomassie Brilliant Blue staining was performed; proteins with similar weight and purity were found to be... Figure 5 ).

[0348] Both wild-type fHbp ID 1 and E218A / E239A double mutants were inhibited by ELISA (Beernink et al. (2010) Clin Vaccine Immunol 17:1074-8). ELISA plates were coated overnight at 4°C with 2 μg / ml recombinant fHbp (ID 1). After blocking with PBS / 1% BSA, mouse anti-fHbp MAb (0.5 μg / ml JAR 1 or JAR 4; 0.1 μg / ml mAb 502 or JAR 5) was premixed with a soluble recombinant protein inhibitor diluted five times at an initial concentration of 50 μg / ml (final concentration) and added to each well of the ELISA plate. The plates were incubated at 37°C for 1 h. MAb binding was detected by incubation at room temperature for 1 h using alkaline phosphatase-conjugated goat anti-mouse IgG (1:10,000; Sigma-Aldrich). As described in Example 1, the ELISA plate was developed. The results showed that, compared to wild-type fHbp, the E218A / E239A double mutant contained four epitopes recognized by anti-fHbp MAbs. The ability of either the soluble mutant or wild-type protein to inhibit the binding of each mAb to wild-type fHbp adsorbed in each well of the ELISA plate was used to determine the epitopes. Figure 6 ).

[0349] The thermal stability of fHbp was also determined. The purified recombinant protein was dialyzed overnight at 4°C using PBS (Roche Applied Science, Indianapolis, IN). The concentration was determined using UV absorbance at 280 nm, and the molar extinction coefficient was 0.8940 M. -1 cm -1 The concentration was adjusted to 0.5 mg / ml. The Degassed protein solution and PBS were loaded into the sample and control wells of a VP-DSC microcalorimeter (MicroCal, Northampton, MA), respectively. The heating rate was 60 °C / h, using an intermediate gain setting. Baseline correction was performed using a buffer-to-buffered scan, and data were normalized to protein concentration. The E218A / E239A double mutant exhibited similar thermostability to the wild-type protein (Figure 7A), as did the R41S mutant. Figure 7B ).

[0350] The immunogenicity of fHbp ID 1 wild-type (WT) and E218A / E239A mutant vaccines was determined in four studies using mice (Studies 3–6). In Study 3, CD-1 mice were immunized with three doses of recombinant WT or mutant fHbp adsorbed with Freund's adjuvant (FA) or aluminum hydroxide (Al(OH)3); in Study 4, CD-1 mice were immunized with one dose of wild-type (WT) or mutant fHbp adsorbed with aluminum hydroxide (Al(OH)3); and in Studies 5 and 6, BALB / c mice were immunized with three doses of WT or mutant fHbp adsorbed with aluminum hydroxide (Al(OH)3). In Study 3, serum was mixed (three mixtures per vaccine group, each mixture derived from serum of 3 to 4 mice). In Studies 4, 5, and 6, individual serum samples were tested (N = 7 to 9 mice per vaccine group).

[0351] To detect the titer of serum anti-fHbp IgG, ELISA plates were activated overnight at 4°C using 2 μg / ml recombinant fHbp ID 1. After blocking with PBS / 1% BSA, mouse antiserum dilutions (diluted five-fold starting at 1:100) were added to each well of the ELISA plate, and the plates were incubated at 37°C for 1 h. The bound anti-fHbp antibody was detected by incubation at room temperature for 1 h using goat anti-mouse IgG (1:10,000; Sigma-Aldrich, St. Louis, MO). The plates were then incubated with p-nitrophenyl phosphate substrate (Sigma-Aldrich, St. Louis, MO) at room temperature for 30 min to develop color, and the optical density was measured at 405 nm.

[0352] like Figure 7C and 7D As shown, in all four studies (studies 3, 4, and 5), Figure 7C Study 6 Figure 7D Compared with the control wild-type fHbp vaccine, serum IgG anti-fHbp antibody responses were reduced in E218A / E239A double mutants in conventional BALB / c or CD-1 mice. In studies 4, 5, and 6, the differences were statistically significant (P<0.05) when individual serum samples were used instead of the mixed serum samples used in study 3. In study 6 (… Figure 7D It also includes an fHbp mutant vaccine with a single amino acid substitution for E239A, the IgG titer of which is lower than that of the WT fHbp vaccine (p = 0.05).

[0353] Table 2 below summarizes the serum bactericidal antibody responses to the E218A / E239A mutant vaccine tested against group B strain H44 / 76. In all studies, the mutant vaccine elicited lower serum bactericidal titers. The differences in studies 4, 5, and 6 were statistically significant (P < 0.05).

[0354] In these studies, neither the double-mutant nor the wild-type fHbp bound to mouse fH. Nevertheless, the lower immunogenicity of the E218A / E239A mutant suggests that the introduction of the double mutation interferes with an epitope crucial for inducing protective bactericidal antibodies. Therefore, eliminating fH binding in the E218A / E239A mutation may not necessarily maintain the optimal structural characteristics for inducing protective antibodies.

[0355] Table 2. Complement-mediated serum bactericidal antibody response in wild-type mice immunized with the fHbp E218A / E239A mutant vaccine.

[0356]

[0357] Example 4: Make F H-binding reduced natural F H BP Identification of variants.

[0358] In studies investigating the binding of naturally occurring fHbp variants to fH in subfamily B (Fletcher et al. (2004) Infect Immun72:2088-2100), also known as variant group 1 (Masignani et al. (2003), as described above), recombinant proteins of the two fHbp variants, ID 14 and 15, showed significantly lower concentration-dependent fH binding than fHbp protein ID 1. Figure 8 Conversely, fHbp ID 14 showed the expected concentration-dependent binding to anti-fHbp MAb JAR 4 and JAR 5, and fHbp ID 15 showed the expected binding to anti-fHbp mAb JAR 5 but not JAR 4 (note that fHbp ID 15 is not expected to bind to JAR 4 because the protein lacks this epitope) (Beernink et al. (2009) Mol Immunol 46:1647-1653; Pajon et al. (2009) Vaccine 28:2122-2129).

[0359] Previous data have shown that the binding of fHbp to fH is similar for variants 1, 2, or 3 (Shaughnessy J et al. (2009) Infect Immun 77:2094-103). Thus, two naturally occurring fHbp variants (such as...) Figure 8The reduction in the binding of fH to fH (shown as fHbpID 14 and 15) was unexpected.

[0360] Figure 8 The data revealed that these two naturally occurring fHbp variants exhibit lower fH binding. The data showed that the amino acid residues contributing to this reduced fH binding could be identified by comparing the fHbp amino acid sequences of the high- and low-binding fH proteins. This strategy differs from targeting conserved residues such as E218A and E239A.

[0361] The fHbp variants can be further classified based on different combinations of the five variable fragments, all of which are derived from one of two genetic lineages named α- or β-type (Pajon et al. (2009) Vaccine28:2122; Beernink and Granoff (2009) Microbiology 155:2873-83). The fHbp ID1, with high fH binding, and ID14, with low fH binding, both originate from module I (all five fragments are α-type). Conversely, another low fH binding protein, fHbp ID15, originates from module IV and is a natural chimera (containing a β-type A fragment and α-type B, C, D, and E fragments). Therefore, the sequence of the natural fH-high binding variant peptide ID28 was used as a control for peptide ID15βA fragment, which contains only the β fragment (module II). See [link to amino acid sequence] for amino acid alignments. Figure 19 Figures A and D are shown below. For the purpose of comparing different variant sequences, the designation fHbp ID 1 is used in this application to refer to specific residues. One of these amino acid residues, the serine (S) at position 41 of the A(β) fragment of peptide 15 (fH low binding), differs from the proline (P) residue of the Aβ fragment of the control peptide 28 (fH high binding). Another amino acid, the E at position 241 of the Eα fragment in both fH low binding variants, differs from the K at position 241 of the fH high binding variant peptide 1.

[0362] Example 5: Make F H-binding reduction at position 41 new mutation F H BP The identification.

[0363] An arginine residue at position 41 (R41) forms a charged hydrogen bond with fH. Figure 9A Replacing arginine with serine eliminates the charged bond (S41, lower right figure). The wells of the ELISA plate were coated with either recombinant WT fHbp ID 1 or R41S mutant ID 1. ELISA results showed that the R41S mutant did not bind to human fH. Figure 10 (See Figure A). The binding of anti-fHbp MAb, JAR 4, and JAR 5 to both the fHbp mutant and wild-type fHbp was almost identical. Figure 10 (Figures B and C). These controls show that the amount of protein adsorbed in each well of the ELISA plate was comparable. Moreover, the R41S mutation does not affect the binding of MAb, and this mutation is in the same domain as the fHbp conformational epitope recognized by JAR 4MAb, and is very close to it (Beernink PT (2009) Mol Immunol 46:1647-53). Another mutation, R41A, which replaces alanine with arginine in fHbp ID 1, also does not bind to fH ( Figure 10 (See Figure A). Therefore, the substitution of non-serine at position 41 can also reduce the binding with fH.

[0364] In surface plasmon resonance experiments, human fH (2400 response units) was immobilized on a CM5 chip (GE Healthcare, Piscataway, NJ) via amino coupling, and the binding of soluble fHbp was detected. The R41S mutant protein (0.5 μM) did not bind to fH (-0.6 response units), while the 0.5 μM wild-type fHbp antigen bound for +22.5 response units, independently confirming the ELISA results. The R41S mutant protein also exhibited greater thermostability compared to wild-type fHbp. Figure 7B ).

[0365] When the mutation is introduced into other fHbp sequence variants in variant group 1 of module group I, the R41S mutation also eliminates fH binding. These variants include fHbp IDs 4, 9, and 74. Figure 11 (See figures A, C, and E). However, in three sequence variants of variant group 2 (module group III or VI), the R41S mutation does not reduce fH binding. These variants include fHbp IDs 19, 22, and 77 (…). Figure 12 (These are small figures A, C, and E, respectively).

[0366] Example 6: In wild-type mice F H BP Immunogenicity of R41S mutant.

[0367] In wild-type mice, the R41S mutant fHbp (ID 1) vaccine elicited a similar serological bactericidal antibody response to the wild-type vaccine (Table 3, below, Studies 2 and 6). In Study 6, the double mutant fHbp vaccine E218A / E239A was used as a negative control, which had previously been reported not to bind to fH (Schneider MC et al. (2009) Nature 458:890-3), but showed attenuated immunogenicity in WT mice (Beernink et al. (2010) Clin Vaccine Immunol 17:1074). The vaccine elicited a significantly reduced bactericidal titer (Table 3, Study 6), thus confirming the data already described in Table 2 above, which shows a reduced antibody response to the E218A / E239A vaccine, as the mutant molecule may have lost an epitope or exhibited minor destabilization (Beernink et al. (2010) Clin Vaccine Immunol 17:1074-8). Conversely, the normal antibody response to the R41S mutant fHbp vaccine indicates that the substitution of arginine with serine did not reduce immunogenicity in mouse models where fH does not bind to either the mutant or wild-type fHbp vaccine.

[0368] Table 3. Complement-mediated serum bactericidal antibody response in wild-type mice immunized with the fHbp recombinant fHbp vaccine.

[0369]

[0370]

[0371] The WT fHbp vaccine binds to human fH; the R41S mutant and the previously described E218A / E239 mutant (Schneideret al. (2009) Nature 458:890-3) do not bind to human fH. In WT mice, natural fH does not bind to either of these vaccines. Figure 1 (Small picture D). a,b P = 0.62; c,d P = 0.01; d,i P = 0.92, T-test (two-tailed).

[0372] Example 7: via R41S mutant F H BP Serum bactericidal antibody response in transgenic mice immunized with the vaccine.

[0373] Serum bactericidal antibody responses in human fH transgenic mice immunized with the R41S mutant vaccine (which does not bind to human fH) were ~3-fold higher than those in human fH transgenic mice immunized with the control wild-type fHbp vaccine (which does bind to fH) (Study 2, Table 4 below). When the data were stratified using serum human fH concentration, mice with fH concentrations <250 μg / ml showed responses similar to those of the mutant and wild-type vaccines. However, in mice with human fH concentrations >250 μg / ml, bactericidal antibody responses in mice immunized with the R41S mutant vaccine were 10-fold higher than those in mice immunized with the wild-type fHbp vaccine (which binds to human fH) (P < 0.05; Table 4).

[0374] Table 4. Serum bactericidal antibody response in human fH transgenic mice immunized with R41S mutant vaccine.

[0375]

[0376] The WT fHbp vaccine binds to human fH; the R41S mutant does not bind to human fH. Data on fH transgenic mice immunized with the wild-type vaccine are shown in Study 2, Table 1 above. a,b Analysis using a general linear regression model showed that different serum human fH concentrations of the fHbp R41S mutant or wild-type vaccine type had different effects on bactericidal titers (P = 0.018). c , d P>0.5. e, f P = 0.05.

[0377] In immunized human fH transgenic mice, there was no significant correlation between serum bactericidal antibody response to the human fH mutant fHbp vaccine that does not bind to human fH and serum human fH concentration. Figure 3 (Small figure B; r = +0.17; P = 0.58), however, as mentioned above ( Figure 3 (Figure A) In transgenic mice, the bactericidal titer induced by the wild-type vaccine bound to fH was negatively correlated with that of the wild-type vaccine (r = -0.65; P = 0.02). The correlation coefficients between the two vaccines were significantly different (P = 0.03).

[0378] A general linear regression model was used to confirm whether the type of fHbp vaccine (fHbp wild-type or R41S mutant) or the concentration of serum human fH affected the serum bactericidal antibody response in transgenic mice. A significant interaction was found between the type of fHbp vaccine and the concentration of human fH with the bactericidal response (likelihood ratio test, P = 0.018). Based on the regression model, the ratio of the inverse of the serum bactericidal GMT in transgenic mice immunized with the R41S mutant vaccine to that in transgenic mice immunized with the human fH-binding fHbp vaccine was estimated at different serum human fH concentrations. Figure 3 (See Figure C). When serum human fH concentration was low (<250 μg / ml), there was no significant difference in bactericidal response. However, when serum fH concentration was high (fH>250 μg / ml, P<0.05; fH>316 μg / ml, P<0.01), the bactericidal response to the R41S mutant vaccine was significantly increased. Since most people's fH concentration falls within this range (…),… Figure 1 (See Figure B). Therefore, the results in transgenic mice suggest that the mutant fHbp molecule that does not bind to fH may be a very good vaccine in humans.

[0379] The experimental protocols and results of the different immune studies mentioned above are summarized in the table below.

[0380] Table 5. Summary of immune studies conducted in human fH transgenic mice.

[0381]

[0382]

[0383] Figure 2 Small figure D provides schematic diagrams of various experimental protocols corresponding to different studies in this application. Figure 2 The numbers above each diagram in small figure D correspond to the superscripts in the table above. Figure 2 In small figure D, group C PS-CRM conjugate refers to the conjugate of meningococcal group C polysaccharide (PS) and cross-reactive mutant diphtheria toxin (CRM), and is called MenC-CRM.

[0384] Example 8: via R41S mutant F H BP The antibody profile of transgenic mice immunized with the vaccine preferentially and closely related to F H binding site Point-based epitope binding.

[0385] The importance of human fH covering the fHbp epitope in the antibody-initiating process with protective activity was investigated. The ability of endogenous human fH in a 1:100 dilution of transgenic mouse serum to bind to fHbp was assessed using ELISA. Consistent with expectations, in the absence of serum anti-fHbp antibodies, the binding of human fH in pre-immunization serum was close to ( ) in both vaccine groups and the control transgenic (Tg) mouse group treated only with aluminum hydroxide. Figure 13 (Figure A). No binding was observed in control WT mice given aluminum hydroxide because native fH does not bind to fHbp. Following vaccination, the amount of “free” human fH detected in the serum of mice immunized with the R41S mutant fHbp was less than that in the serum of mice immunized with a vaccine binding to human fH (P = 0.001). Figure 13 (Figure B), or serum from transgenic mice given only aluminum hydroxide ( Figure 13 (See Figure B). Because the IgG titers of anti-fHbp antibodies were similar in both fHbp vaccine groups (…). Figure 13 (Figure D) The detectable concentration of human fH in serum after R41S immunization was low. The ability of anti-fHbp antibody to inhibit the binding of human fH to fHbp was higher than that induced by the wild-type vaccine binding human fH, which is consistent with the previous observation. Different dilutions of serum from each mouse were also tested in the presence of 5% normal human serum as the fH source (N=11 per group). In the 1:100 and 1:400 dilutions, the inhibitory effect of the R41S mutant vaccine group was significantly increased (P<0.03). Figure 13 (See Figure C). In summary, the increased fH inhibition in the R41S mutant vaccine group suggests a difference in the antibody profiles induced by the two vaccines. For example, antibodies induced by the mutant fHbp vaccine may act more on epitopes near the fH binding site, and these antibodies may block fH binding more effectively than those induced by the fH-binding vaccine. Furthermore, when antibodies target the fH-binding surface-exposed regions of fHbp, they are expected to exhibit stronger functional bactericidal activity.

[0386] A significant correlation was also observed between the ability of each mouse serum to inhibit the binding of human fH to fHbp and the reciprocal of the serum bactericidal titer (Spearman r value, 0.69, and P value 0.0004). Figure 13 (Figure E). In the serum bactericidal response, the reduced binding of the complement inhibitor fH to the bacterial surface of the test organism may have contributed to the increased bactericidal titer induced by the mutant fHbp vaccine. Therefore, the results of predicting protective antibody activity by the ability of anti-fHbp antibodies to inhibit fH binding showed that the R41S vaccine was better.

[0387] Example 9: F H binding is reduced F H BP Identification of other mutants in ID 1.

[0388] Position 241 is located on the fH-binding surface of fHbp ID 1. The effect of amino acid substitutions at residue 241 of the fHbp ID 1 sequence on fH binding was investigated. Figure 14As shown in inset A, the substitution of lysine (K) residue 241 with glutamic acid (E) in fHbp ID 1 (K241E) does not affect fH binding. Upon reverse substitution, the E241K mutant in module group IV at fHbp ID 15... Figure 14 (Figure C) showed no significant effect on the binding of fH compared to wild-type fHbp (<2 times); Figure 14 (See Figure C). (Amino acid residue numbering is based on the sequence of fHbp ID 1).

[0389] In fHbp ID 1, mutations are found at positions R41, H119, R130, and K241. Production of the fHbp mutants is as described above. The binding of the R41A, H119A, R130A, and K241E single-substitution mutants to human fH, as well as to MAb, was then evaluated.

[0390] like Figure 10 As shown in small figure A, R41S and R41A substitutions in fHbp ID 1 reduce the binding to human fH. Figure 10 As shown in insets B and C, the R41S and R41A mutants retain binding to MAb JAR 4 and JAR 5, respectively, indicating that these epitopes are preserved in the R41S and R41A mutants.

[0391] like Figure 15 As shown in small figure A, the substitution of H119A and R130A in fHbp ID 1 reduces the binding to human fH. Figure 15 As shown, compared to the corresponding wild-type fHbp ID 1 (inset C), the R130A mutant retains binding to MAb JAR5 (inset B), but binding to MAb JAR4 is reduced. These data indicate that, after amino acid substitution, the JAR5 epitope is retained in both the H119A and R130A mutants; the JAR4 epitope is partially retained.

[0392] Example 10: F H BP Module group IV F H BP Mutants of sequence variants.

[0393] For fHbp sequences classified as module group IV variant 1 ( Figure 16 Regarding "V" in variant group 1 A "The fragment is derived from the genetic lineage (β), which is different from the corresponding "V" in variant group 1 of module group I fHbp sequence variants." A The fragment is named the α fragment (Beernink et al. (2009) Microbiology 155:2873). According to the nomenclature used on the website pubmlst.org / neisseria / fHbp / , the α and β lineages can also be named lineage 1 and 2, respectively.

[0394] In the fHbp amino acid sequence variant of module group IV, position 41 is typically serine, not arginine. In the mutant fHbp ID 15 (module group IV), the substitution of serine with proline (S41P) eliminates the binding to fH. Figure 17 Control proteins included recombinant fHbp ID 1 and 28 (native fH high-binding proteins) and fHbp ID 15 (native fH low-binding proteins). As described above, the binding of human factor H or anti-fHbp MAb to fHbp was detected using ELISA. The binding of anti-fHbp MAb JAR5 to WT fHbp ID 1 and 15, and the S41P mutant of fHbp ID 15, was similar. Figure 17 (See small figure B). JAR 31 shows the expected combination with fHbp ID 28 ( Figure 17 (Small image C).

[0395] Example 11: Module Groups III and VI F H BP The substitution of the R41S amino acid in the sequence variant does not affect F The binding of H.

[0396] All fHbp sequence variants classified as variant 2 are natural chimeras, containing fragments derived from the α and β lineages. Figure 16 Specifically, in variant 2 protein, the "V" A "The fragment is derived from the α lineage and, like module group I, typically contains arginine at position 41 (residue numbering based on fHbp ID 1). Although it has been found that R41S in all module group I proteins eliminates fH binding ( Figure 11 and 10 And Table 6), but the R41S mutations in fHbp ID19, 22, and 77 in variant group 2 (module group III or VI) do not eliminate fH binding (and Table 6), Figure 12 (See small figures A, C, and E, and Table 7).

[0397] Example 12: Non-binding human F Synthesis of H F H BP Chimeric protein.

[0398] Figure 16 The last module diagram is fHbp chimera I (Beernink and Granoff (2008) Infect. Immun. 76:2568-75). The fusion node of fHbp ID 1 part (variant 1, module group I) and fHbp 77 part (variant 2, module group VI) is at V. C G136 in the clip. Figure 16 In the process, the V in the chimeric protein C The half-gray and half-white representation indicates that the fragment is a fusion of α-lineage and β-lineage sequences. When R41S is substituted and introduced into the variant 2fHbp protein, its binding to fH is unaffected. Figure 12 (See figures A, C, and E). Conversely, when R41S was substituted into the fHbp chimeric I protein, the mutation eliminated the binding of fH (…). Figure 18 This result was unexpected because the V of chimeric I and fHbp ID 77... A The amino acid difference between the fragments is only one amino acid residue (Gly30 in the chimeric antigen, not Ser30); Figure 19 ). In V C In the fragment, eight residues between positions 98 and 135 are different (Figure 19), which may explain why the R41S mutation eliminates fH binding in the chimeric protein but not in the native variant 2 protein (see [link to original text]). Figure 16 The diagram is shown in the image; the complete amino acid sequence is shown in the image. Figure 19 (Figure A). These observations suggest that, for fHbp in variant 2, the residues at this position in the Vc region have a significant impact on the stability of the fHbp-fH complex.

[0399] Example 13: F H BP Other amino acid substitutions in ID 77 (module group VI) F The effect of H binding.

[0400] Alanine mutations at positions K113, K119, and D121 were introduced into fHbp 77 (module group VI, antigen variant group 2). As shown above, residue position numbering was based on fHbp ID 1. fHbp was generated according to the description in the Materials and Methods section above.

[0401] As described in Example 2 above, the ability of these mutants to bind to human fH was detected using ELISA and compared with the corresponding wild-type fHbp. Compared with wild-type fHbp ID 77, the introduction of the K119A mutation increased fH binding by approximately 4-fold. Figure 20 (See small figure A); K113A has no effect on fH binding ( Figure 20 (See Figure A), and compared to the binding of wild-type fHbp ID 77 to fH, D121A reduced fH binding by approximately 4 times (Figure A). Figure 20 (See Figure A). Anti-fHbp JAR 31 bound to all three mutants, indicating that amino acid substitutions had no effect on the epitopes recognized by this mAb.

[0402] In fHbp ID 77, dual amino acid substitutions R41S / K113A, R41S / K119A, and R41S / D121A were also constructed. ELISA results showed that, compared to wild-type fHbp ID 77, the R41S / K119A mutant reduced fH binding by approximately 5-fold. Figure 21 (See Figure A), while the R41S / K113A and R41S / D121 mutants reduce fH binding by approximately 10-fold ( Figure 21(See Figure A and Table 6). Anti-fHbp mAb JAR 31 showed similar binding to all three double mutants of ID 77 and wild-type fHbp ID 77, indicating that the amounts of all fHbp variants adsorbed in the wells of the ELISA plate were similar and that these amino acid substitutions had no effect on the epitopes recognized by this mAb.

[0403] A triamino acid substitution R41S / K113A / D121A was introduced in fHbp ID 77. This tri-mutant does not bind to fH. Figure 22 (See Figure A). The mutant fHbp maintained its binding to JAR 31 ( Figure 22 (small figure C), but eliminated the binding with JAR4 ( Figure 22 (See Figure B). Conversely, the K113A / D121A double mutant reduced fH binding by approximately 10-fold, indicating that these substitutions, in addition to R41S substitution, also contributed to the loss of fH binding.

[0404] Example 14: F H BP Amino acid substitution pairs in ID 22 (module group III) F The effect of H binding.

[0405] fHbp ID 22 is a representative fHbp sequence variant in module group III (variant group 2, Figure 16 Mutations were introduced at position 22 of fHbp ID: R80, D211, E218, D248, G236 (Table 6) and R41, Q38, A235, Q126, D201, and E202 (Table 7). The fHbp mutation was produced as described above. Specifically, substitutions of R80A, D211A, E218A, E248A, R41S, Q38A, Q126A, G236I, A235G, D201A, and E202A were introduced individually at fHbp ID 22. Additionally, a T220A / H222A double substitution was introduced at fHbp ID 22. The binding of the mutant to fH and its binding to anti-fHbp mAb were then characterized using ELISA. Figure 6 and Figures 7A-7D ).

[0406] As described in Example 2 above, the ability of these mutants to bind to human fH was tested and compared with the ability of wild-type fHbp ID 22 to bind to human fH. Results are shown below. Figure 23 The smaller graphs are AC, and summarized in Tables 6 and 7. (See table 7 for details.) Figure 23 As shown in insets A and B, compared to the binding of wild-type fHbp ID 22 to fH, the substitution of D211A, R80A, E218A, and E248A in fHbp ID 22 reduces its binding to fH by more than 50-fold (see also Table 6). Figure 23As shown in small figure C, substitution with R41S, Q38A, and Q126A does not significantly reduce binding to fH (<4-fold, see also Table 7).

[0407] like Figure 24 As shown in insets A and B, the R80A, D211A, E218A, and E248A mutants of fHbp ID 22 maintained their binding to MAb JAR31, indicating that these mutants retained the JAR31 epitope.

[0408] like Figure 25 As shown in insets A and B, the D211A and E218A mutants of fHbp ID 22 maintained binding to MAb JAR4, indicating that these mutants retained the JAR4 epitope. Figure 25 As shown, the R80A mutant did not retain binding to MAb JAR4 (inset A), while the E248A mutant showed reduced binding to MAb JAR4 (inset B).

[0409] like Figure 26 As shown in small figures A and B, the R80A, D211A, E218A, and E248A mutants of fHbp ID 22 maintained their binding to MAb JAR35, indicating that these mutations all preserved the JAR35 epitope.

[0410] like Figure 27 As shown in small figure A, compared with the binding of wild-type fHbp ID 22 to human fH, the T220A / H222A disubstituted and G236I monosubstituted fHbp ID 22 reduces its binding to human fH by more than 50-fold (see also Table 6). Figure 27 As shown, the T220A / H222A mutant with fHbp ID 22 maintains binding to MAb JAR31 (small figure B), MAb JAR 35 (small figure C), and MAb JAR 4 (small figure D), indicating that the T220A / H222A mutant retains the JAR31, JAR35, and JAR4 epitopes. Figure 27 As shown, the G236I mutant in fHbp ID 22 maintained binding to MAb JAR 35 (inset C), but binding to MAb JAR 31 was reduced (inset B), and binding to JAR 4 was less or non-existent (inset D).

[0411] like Figure 28 , Figure 4 As shown, the R41S, Q38A, and A235G monosubstituted compounds of fHbp ID 22 do not significantly reduce binding to human fH. Figure 28As shown, the R41S, Q38A, and A235G mutants maintained their binding to MAb JAR31 (inset B) and MAb JAR35 (inset C), indicating that the epitopes of JAR31 and JAR35 were preserved in the R41S, Q38A, and A235G mutants.

[0412] like Figure 29 As shown in small figure A, the monosubstituted Q126A, D201A, and E202A substitutions of fHbp ID 22 do not significantly reduce the binding to human fH. Figure 29 As shown in small figure B, the Q126A, D201A, and E202A mutants maintained their binding to MAb JAR35, indicating that these mutations all retained the JAR35 epitope.

[0413] The effects of single or double amino acid substitutions on the binding of fHbp ID 1, ID 22, and ID 77 to human fH, as well as their binding ability to different monoclonal antibodies, are summarized in Tables 6 and 7.

[0414]

[0415]

[0416]

[0417]

[0418] like Figure 30 As shown in small figure A, the binding of the E218A monosubstituted fHbp ID28 to human fH is lower than that of wild-type fHbp ID28 to fH. Similarly... Figure 30 As shown in small figure A, the monosubstitution of E197A, K245A, and D201A in fHbp ID 28 did not significantly reduce the binding with fH. Figure 30 Figure B shows the binding of mouse polyclonal anti-fHbp ID28 antiserum to different proteins (WT fHbp; and single substitutions of fHbp ID28 for E197A, K245A, and D201A). Figure 30 The data in small figure B shows that the amounts of different fHbp in the ELISA plate are similar. Figure 30 As shown in insets C and D, the E218A mutant binds to JAR31 and JAR33 MAb, indicating that it retains the full configuration of the epitopes that these MAb can recognize.

[0419] The overall immunogenicity of the fHbp mutant vaccine, administered to wild-type mice where the native fH does not bind to the mutant or wild-type vaccine, can be determined. Data generated by this model can provide an overall assessment of whether the mutant vaccine retains epitopes crucial for evoking serum bactericidal antibodies. For example, the E218A / E239A mutant of fHbp ID 1 eliminates binding to human fH, but has been found in multiple studies to elicit a diminished bactericidal antibody response in WT mice (Table 2 above). Immunogenicity assays were performed as described in Example 1 above. Titers of IgG and bactericidal antibodies were measured and compared to corresponding levels in mice administered the appropriate wild-type and / or negative control. If the mutant vaccine retains the key epitopes required to elicit bactericidal activity, the antibody levels induced in wild-type mice are not expected to differ significantly from those induced by the appropriate wild-type fHbp vaccine.

[0420] Example 15: F H BP Variant-induced bactericidal response.

[0421] Wild-type BALB / c mice (whose fH does not bind to WT fHbp) were immunized with three doses of recombinant fHbp vaccine administered intraperitoneally, three weeks apart. Each dose contained 10 μg of recombinant fHbp and 300 μg of Al(OH)3 in a 100 μl volume (final buffer composition: 10 mM histidine, 150 mM NaCl, pH 6.5). Three weeks after the third dose, blood samples were collected via cardiac biopsy.

[0422] Serum bactericidal titers against group B strain H44 / 76 (ID 1) or group W-135 strain Ghana 04 / 07 (ID 22) were determined using human complement with IgG removed (Beernink et al, J Immunology 2011). No difference was defined as no significant difference in the geometric mean titer (GMT) between the mutant and each WT vaccine (T-test of log10 converted titers, P > 0.10).

[0423] See data Figures 31-33 . Figure 31 Serum bactericidal titers in mice immunized with the fHbp ID 1 mutant vaccine, which reduces binding to human fH, are shown. Symbols represent individual titers in mice tested against group B strain H44 / 76 (ID 1). Horizontal lines represent the geometric mean titer. There were no significant differences in GMTs induced by the mutant vaccines compared to those induced by the WT fHbp ID 1 vaccine (P>0.10).

[0424] Figure 32The serum bactericidal titers of mice immunized with the mutant fHbp ID 22 vaccine, which showed reduced binding to human fH, are shown. Each symbol represents the bactericidal titer of individual mice tested against group W-135 strain Ghana 7 / 04 (ID 22). The horizontal line represents the geometric mean titer. In the top figure, there was no significant difference in GMT between the mutant vaccines (D211A, E218A, E248A, and T220A / H222A) and the WT ID 22 vaccine (P>0.10). In the bottom figure, the GMT induced by the mutant vaccines (R80A and G236I) was significantly lower than that induced by the WT ID 22 vaccine (P<0.05).

[0425] Figure 33 Serum bactericidal titers of mice immunized with the mutant fHbp ID 77 vaccine, which showed reduced binding to human fH, are shown. Symbols represent the bactericidal titers of individual mice tested against group W-135 strain Ghana 7 / 04 (ID 22). Horizontal lines represent the geometric mean titer. GMT levels in mice immunized with the R41S / K113A / D121A triple mutant ID 77 vaccine were significantly lower than those in mice immunized with the WT vaccine (P<0.05).

[0426] Table 8 summarizes... Figures 31-33 The immunogenicity data are shown. No difference means there is no significant difference in the geometric mean titer (GMT) between the mutant and each WT vaccine (T-test of titers converted to log10, P>0.10).

[0427] Table 8. Immunogenicity of fHbp mutants with reduced fH binding.

[0428]

[0429]

[0430] Example 16: Sequence alignment.

[0431] Figure 34 and Figure 35Amino acid sequence alignments are provided for fHbp ID 1 (SEQ ID NO:1), fHbp ID 22 (SEQ ID NO:2), fHbp ID 77 (SEQ ID NO:4), fHbp ID 28 (SEQ ID NO:3), and the ID1 / ID77 chimera (SEQ ID NO:8). ID 28 is the control sequence for fHbp variant group 3. As described by Schneider et al. ((2009) Nature 458:890-3), the H-factor binding interface residues (highlighted in gray) are described as hydrogen bonds or ion-to-ion interactions. GEHT (SEQ ID NO:27) at positions 136 to 139 represents the node between ID1 and ID77 of the chimeric fHbp.

[0432] Figure 35 The amino acid sequences of fHbp ID 1 (SEQ ID NO:1), fHbp ID 22 (SEQ ID NO:2), fHbp ID 77 (SEQ ID NO:4), fHbp ID 28 (SEQ ID NO:3), and the ID1 / ID77 chimera (SEQ ID NO:8) were compared. Residues marked in gray indicate mutated residues, and these are summarized in Table 7.

[0433] Table 9 below summarizes the MAb reactivity of fHbp ID 1, ID 22, ID 77, and ID 28.

[0434] Table 9

[0435]

[0436] Example 17: F H / F H BP The correlation between inhibitory efficacy and bactericidal activity; and N SP Function A.

[0437] Materials and methods

[0438] Mouse anti-fHbp mAbs. Hybridoma cell lines producing mouse fHbp-specific monoclonal antibodies (mAbs) JAR 3 (IgG3), JAR 5 (IgG2b), and mAb502 (IgG2a; Giuliani et al. (2005) Infect. Immun. 73:1151; and Scarselli et al. (2009) J. Mol. Biol. 386:97) were used. Control mAbs included SEAM12 (Granoff et al. (1998) J. Immunol. 160:5028), which reacts with the group B capsule, and anti-PorA P1.7 (NIBSC code 01 / 514, from the National Institute for Biostandards and Reference Materials, Potters Bar, United Kingdom).

[0439] Human IgG1 chimeric mouse anti-fHbp mAb. RNA isolated from hybridoma cells was converted to cDNA using the Omniscript RT kit (Qiagen) according to the manufacturer's instructions. The cDNA was amplified using immunoglobulin heavy chain (H) and light chain (L) specific primers (Wang et al. (2000) Infect. Immun. 68:1871) and inserted into a pGem vector (Promega) for sequencing. Based on the sequenced data, specific primers were designed to facilitate the insertion of mouse VH and VL sequences into a modified FRT bicis-reverse eukaryotic expression vector (Invitrogen). For each antibody, the mouse VL sequence was inserted downstream of the human κL chain leader sequence and within the frame of the human κL chain constant sequence. The mouse VH sequence was inserted downstream of the human H chain leader sequence and within the frame of the complete human IgG1 constant region sequence. The vector utilized an internal ribosome entry fragment (IRES) between the VH and VL sequences to facilitate balanced translation of both chains. The DNA sequences of all constructs were validated prior to transfection.

[0440] Flp-In CHO cells (Invitrogen) were seeded into each well of a Nunclon Δ6-well plate (containing 2 mL of Flp-In medium), at a density of 3.5 x 10⁶ cells per well. 5Cells were cultured overnight at 37°C and 5% CO2. Once the cells reached 80% confluence, they were transfected using TransFast transfection reagent (Promega) with pOG44 and an FRT vector containing VH and VL intercalations (9:1 ratio). Forty-eight hours after transfection, the cells were trypsinized and placed in new 6-well plates for drug selection using 600 μg / ml hygromycin. Transfected cells were cultured in serum-free suspension on Excel 302 medium (Sigma Aldrich) for approximately two weeks. Antibodies from the cell culture supernatant were concentrated using a 200 ml agitator (Amicon) and under nitrogen pressure before purification. The antibodies were purified using a HiTrap Protein G column (GE Healthcare) followed by extensive dialysis with PBS. BSA was added to a final concentration of 1%, and the cells were aliquoted and stored at -30°C.

[0441] ELISA. The concentration of human IgG mouse chimeric mAb was determined using a capture ELISA assay via goat anti-human κ chain specific antibody (Biosource) bound to each well of the ELISA plate. The bound human IgG was detected using a goat anti-human IgG antibody (Invitrogen) conjugated with alkaline phosphatase. Antibody concentration was determined by comparing the concentration-dependent binding with a human IgG1 standard (a monoclonal κ chain antibody derived from human myeloma, Sigma). The binding of anti-fHbp mAb to fHbp was measured using an ELISA assay via recombinant fbp on the ELISA plate. The secondary detection antibody was a goat anti-human κ chain specific antibody (Biosource) conjugated with alkaline phosphatase.

[0442] Surface plasmon resonance (SPR). Recombinant fHbp (500 or 1000 response units) were immobilized on a CM5 chip (GE Healthcare, Piscataway, NJ) using an amine coupling kit (GE Healthcare). SPR was then used to determine the binding kinetics of human-mouse chimeric mAbs to fHbp. Binding kinetics were determined using a Biacore T / 100 instrument (GE Healthcare, Piscataway, NJ) at mAb concentrations ranging from 0.016 to 50 μg / ml (0.1 μM to 333 μM).

[0443] Binding to Neisseria meningitidis was determined by flow cytometry. The binding of the chimeric mAb to the surface of live encapsulated bacteria was detected using strain H44 / 76 (B:15:P1.7,16; ST-32), a strain with a relatively high expression of fHbp ID1 (Welsch et al. (2008) J. Infect. Dis. 197:1053; Welsch et al. (2004) J. Immunol. 172:5606). In some experiments, H44 / 76 syngeneic knockout (KO) mutants were detected, which did not express fHbp, NspA, or both proteins. Genotypes included fHbp:Erm, NspA:Spc, and fHbp:Erm / NspA:Spc (Lewis et al. (2010) PLoS Pathog. 6:e1001027). Binding assays were performed as previously described, but the test and control antibodies were co-incubated with cells at room temperature for 1 hour, instead of 2 hours on ice. Antibodies binding to bacteria were detected using CF488A goat anti-human IgG (BioTium).

[0444] Inhibition of fH binding. The ability of anti-fHbp mAb to inhibit the binding of fH to fHbp was detected using ELISA. Recombinant fHbp (2 μg / ml) was coated onto each well of the ELISA plate. mAb dilution was added, and the plate was incubated at 37°C for 2 hours, followed by the addition of 2 μg / ml human fH (Complement Tech.), and incubation at room temperature for another 1 hour. After washing with PBS-0.05% Tween 20, sheep polyclonal antiserum of fH (Abcam) was added, followed by donkey anti-sheep IgG antibody conjugated with alkaline phosphatase (Sigma Aldrich), to detect fH binding. Results are expressed as the percentage of fH binding inhibition in the presence of anti-fHbp mAb compared to the percentage of fH binding inhibition in the absence of mAb.

[0445] Flow cytometry was used to detect the ability of anti-fHbp mAb antibody to inhibit the binding of fH to the surface of live bacterial cells. H44 / 76 bacteria were incubated with 50 μg / ml anti-fHbp mAb and different concentrations of purified fH at room temperature for 30 minutes. After washing the cells, sheep polyclonal antiserum containing fH (Lifespan Bioscience) was added, followed by donkey anti-sheep IgG antibody conjugated with Alexa Fluor 488 green fluorescent dye (Invitrogen) to detect the bound fH. In some experiments, 20% IgG-depleted human serum containing 90 μg / ml fH was used as the source of human fH. To avoid lysis, the human serum was heated at 56°C for 30 minutes to inactivate the heat-sensitive complement components necessary for lysis. This heat treatment did not affect the activity of fH.

[0446] Human complement source. When used to detect bactericidal activity and C4b deposition, the primary complement source is serum from healthy adults with normal total hemolytic complement activity and no detectable serum bactericidal activity against the test strain. To eliminate non-bactericidal IgG antibodies that may promote or inhibit the activity of the test mAb, IgG from the complement source is removed using a protein G column (HiTrap Protein G, GE Life Sciences, Piscataway, NJ). 1 ml of human serum is aliquoted and passed through a PBS-equilibrated protein G column (1 ml HiTrap Protein G, GE Life Sciences, Piscataway, NJ), and the eluent is collected. Adequate IgG removal is monitored using an IgG capture ELISA, and CH50 activity is determined using a commercially available assay (EZ Complement CH50 assay kit, Diamedix Corp., Miami, FL). To avoid lysis when detecting C4b deposition, an anti-human complement C6 affinity column was used to remove complement sources, as described above (Plested and Granoff (2008) Clin. Vaccine Immunol. 15:799). In some experiments, commercially available human complement sources have been deprived of fH or C1q (Complement Tech.) and, as described above, IgG has also been removed.

[0447] Serum bactericidal activity assay. As previously described (Beernink et al. (2009) J. Infect. Dis. 199:1360), bactericidal activity was determined using group B bacteria H44 / 76 in logarithmic phase and 20% human serum with IgG removed as complement source. Before the assay, anti-fHbp mAb was centrifuged at 100,000 x g for two hours to remove polymers. Bactericidal activity (BC) 50％ The mAb concentration is defined as a 50% reduction in CFU / ml compared to the negative control well at time zero, after 60 minutes of incubation with the reaction mixture.

[0448] C1q-dependent C4b deposition on Neisseria meningitidis. Flow cytometry was used to detect the deposition of human C4b on the surface of viable group B strain H44 / 76 bacteria. Bacteria were grown as described in the bactericidal assay section above, and incubated with 5% C1q-depleted human serum (Complement Tech.), which had also been depleted of complement C6 to avoid lysis (see above). Different concentrations of chimeric human-mouse anti-fHbp mAb were added, with or without 20 μg / ml purified C1q protein (Complement Tech.). Human C4b binding to bacteria was detected using an anti-human C4b antibody conjugated with fluorescent isothiocyanate (Meridian LifeScience).

[0449] result

[0450] JAR 3 and JAR 5 mAbs mutually inhibit binding to fHbp and recognize overlapping epitopes containing glycine and lysine at positions 121 and 122, respectively. The epitopes recognized by the two paratopes bind differently in Western blot analysis with different fHbp amino acid sequence variants, thus allowing for differentiation. Murine hybridomas JAR 3 and JAR 5 originate from the same VH and VL germline genes, but their respective CDR regions differ (except for VL CDR2). The VH and VL germline genes encoding mouse mAb502 differ from those of JAR 3 or JAR 5. The conformational epitope recognized by mAb502 requires arginine at position 204 and does not inhibit the binding of JAR 3 or JAR 5 to fHbp. Therefore, the fHbp epitope recognized by mAb502 differs from that recognized by JAR mAb. The GenBank accession numbers for the vL and vH genes of mAb502 are EU835941 and EU835942, respectively. The GenBank accession numbers for the VL and VH regions of the JAR3 and JAR5 antibodies are as follows: JF715928, JAR3 variable heavy chain; JF715929, JAR3 variable light chain; JF715926, JAR5 variable heavy chain; and JF715927, JAR5 variable light chain.

[0451] Figure 36 Based on crystal structure coordinates, a complex model of fHbp and fH fragments (Schneider et al. (2009) Nature 458:890) is presented. In the complex with fHbp, the spatial relationships of amino acid sequences that have been previously reported to affect the binding of anti-fHbpmAb502, JAR 3, and JAR 5 to the fHbp fH fragment are marked in light gray.

[0452] Three human-mouse chimeric anti-fHbp mAbs exhibited similar binding properties. Three human-mouse chimeric anti-fHbp antibodies were constructed, in which the complementary sites of JAR 3, JAR 5, and mAb502 were paired with the constant region of human IgG1. In ELISA, the three mAbs showed similar binding to recombinant fHbp adsorbed in each well of the ELISA plate. Figure 37A Surface plasmon resonance (SPR) analysis showed that the binding kinetics of the three mAbs with 200, 500, or 1000 RU immobilized fHbp were similar, with detection concentrations ranging from 0.016 to 2.25 μg / ml. Representative data for ID 1 of the 0.25 μg / ml (1.7 μM) mAb and the 1000 RU immobilized fHbp are shown in [reference needed]. Figure 37B Although the peak binding rate of mAb502 to fHbp was lower than that of JAR 3 or JAR 5, the binding rates K0 were higher. aSimilar (JAR 3, JAR 5, and mAb 502 are 4.25E+06, 2.26E+06, and 1.19E+06, respectively). The dissociation rates of all three mAbs are slow, making it impossible to calculate the accurate dissociation constant. Ka for each mAb... d The order of magnitude of the value is E-05.

[0453] mAbs bound to the surface of viable group B strain H44 / 76 bacteria were detected by indirect fluorescence flow cytometry. When the mAb concentration was between 0.8 and 40 μg / ml, the binding of the three mAbs was indistinguishable from each other. The binding results at 4 μg / ml are shown in [the table below]. Figure 37C The presence of heat-inactivated 20% deIgG human serum does not affect binding; said serum contains ~90 μg / ml of human fH (comparative). Figure 37D and Figure 37C ).

[0454] Figure 37-37D: Binding of fHbp-specific mAb to fHbp. Figure 37A ELISA. The bound IgG was detected using an alkaline phosphatase-conjugated anti-human κ light chain specific antibody. Figure 37B Surface plasmon resonance. Anti-binding of fHbp mAb (0.25 μg / ml) to immobilized recombinant fHbp (1000 RU). Figure 37C Flow cytometry. Binding of anti-fHbp mAb (4 μg / ml) to live Neisseria meningitidis B strain H44 / 76 cells. JAR 3, black dashed line; JAR 5, gray line; mAb502, black line. An irrelevant mAb (100 μg / ml) was used as a negative control (gray bar graph). The three binding curves of anti-fHbp mAb overlap. Figure 37D Flow cytometry. mAb concentration and Figure 37C The same formula was used, with the addition of 20% human serum (without IgG) as a source of human fH (~90 μg / ml).

[0455] All three human-mouse chimeric mAbs activated the human classical complement pathway, but only JAR 3 and JAR 5 exhibited bactericidal activity. Activation of the classical complement pathway begins when IgG binds to the bacterial surface, with sufficient antigen-antibody complexes to attract C1q proximal to the Fc region of the antibody, which subsequently activates C4b. Detection of C4b binding to the surface of live Neisseria meningitidis strain H44 / 76 was used as an alternative to the detection of C1q binding and C4b activation, and served as a marker of classical complement pathway activation.

[0456] When the complement source is 5% human serum de-C1q and IgG removed, the C4b deposition induced by anti-fHbp mAb is negligible. Figure 38AWhen 20 μg / ml of purified C1q was added to the reaction mixture, each mAb activated C4b deposition. Figure 38B Although the binding of each mAb activates the classical complement pathway, only JAR 3 and JAR 5 possess complement-mediated bactericidal activity. Figure 38C The mean concentration ± SE of 50% sterilization in JAR 3 was 9 ± 0.85 μg / ml in three tests; JAR 5 was 15 ± 2 μg / ml (P = 0.024); and mAb502 was >100 μg / ml (P < 0.0003 compared with JAR 3 or JAR 5).

[0457] Figures 38A-38C C1q-dependent complement activation in capsular group B bacterial strain H44 / 76. Figure 38A Flow cytometry. 4 μg / ml mAb and C1q-removed complement (5% IgG-removed human serum) activated C4b deposition. Anti-fHbp mAb JAR 3, black dashed line; JAR 5, gray line; mAb502, black line; and irrelevant human mAb (100 μg / ml; gray bars) (overlapping data). Figure 38B Flow cytometry. Except for the addition of 20 μg / ml purified C1q protein to the reactants, the symbols and conditions were the same as before. Figure 38A same. Figure 38C Bactericidal activity. The number of bacteria that survived incubation at 37°C for 60 minutes with various mAbs and complement (20% human serum with IgG removed).

[0458] Chimeric mAbs JAR 3 and JAR 5, but not mAb502, inhibited fH binding. In previous studies, mouse mAbs JAR 3 and JAR 5 inhibited fH binding to fHbp, while mouse mAb502 did not. ELISA results showed that human IgG1 chimeric JAR 3 and JAR 5 mAbs also inhibited fH binding to fHbp, while chimeric mAb502 did not inhibit fH binding. Figure 39A When using 20% ​​heat-inactivated, IgG-removed human serum as the fH source, 50 μg / ml of chimeric JAR 3 or JAR 5, instead of mAb502, inhibits the binding of fH to the surface of live bacterial cells. Figure 39B Even concentrations as low as 2 μg / ml of JAR 3 or JAR 5 were able to inhibit fH binding. Figure 39C Although the inhibition was not complete compared to 50 μg / ml mAb (Figure 39B).

[0459] Figures 39A-39C : Inhibition of fH binding by anti-fHbp mAb. Figure 39A ELISA: Using fH (4 μg / ml) with solid-phase recombinant fHbp. Figure 39B and 39C Flow cytometry was performed using live bacterial cells of group B strain H44 / 76; the light gray area represents bacteria and fH (~90 μg / ml) in 20% human serum (without mAb and IgG removed); the solid black line represents bacteria and serum fH + mAb 502, 50 μg / ml; the dashed black line represents bacteria and serum fH + JAR 3, 50 μg / ml; the solid gray line represents bacteria and serum fH + JAR 5, 50 μg / ml; the dark gray area represents bacteria without fH or mAb as a negative control. fH binding was detected using an fH-specific sheep antibody. Figure 39C Except for using 2 μg / ml instead of 50 μg / ml for the detection of each anti-fHbp mAb, the conditions were the same as... Figure 39B same.

[0460] The observed correlation between bactericidal activity and the inhibition of fH binding by mAbs does not necessarily prove that inhibition is the reason why JAR 3 or JAR 5 have higher bactericidal activity than mAb 502. For example, the spatial relationship of the fHbp epitopes on the bacterial surface recognized by anti-fHbp mAbs that inhibit fH binding is different from the spatial relationship of the epitopes recognized by anti-fHbp mAbs that do not inhibit fH binding (compare, for example, the positions of amino acid residues that previously affected mAb 502 binding (Scarselli et al. (2009) J Mol Biol 386:97-108) with the positions of amino acid residues that bound JAR 3 and JAR 5 (Beernink et al. (2008) Infect Immun 76:4232-4240)). Figure 36 These spatial differences may potentially influence the formation of functional membrane attack complexes, independent of fH downregulation.

[0461] To determine whether the differences in epitope positions affecting bactericidal activity depended on the inhibition of fH, anti-fHbp bactericidal activity was detected using complement (20% human serum, also with IgG removed) after fH removal. In the absence of fH, all three mAbs showed similar complement-mediated bactericidal activity (BC). 50％ (1.2 to 1.4 μg / ml, Table 10). Conversely, when purified human fH was added to the reaction mixture, mAb502 no longer exhibited bactericidal properties (BC). 50％>100 μg / ml, Figure 39B). Adding fH to the reaction mixture also reduced the bactericidal activity of the two control mouse mAbs against the group B capsule or PorA (the bactericidal activity of each group B was determined using complement with fH removed). 50％ value( Figure 39C ) and each BC of complement with added fH 50％ value( Figure 39C (Compared), but the effect of adding fH was not as significant as that of anti-fHbp mAb.

[0462] Table 10. Bactericidal activity of anti-fHbp mAb as tested with human complement that has been deprived of fH.

[0463]

[0464]

[0465] Eliminating the binding of fH to NspA increased the bactericidal activity of anti-fHbp mAb JAR 3 and JAR 5, but not mAb502. The concentration of anti-fHbp mAb with fH removed was lower as a lysing agent compared to when fH complement was added, suggesting that the inhibition of fH binding by JAR 3 or JAR 5 is incomplete in the presence of fH (e.g., because fH also binds to another meningococcal ligand such as NspA (Lewis et al. (2010) PLoS Pathog. 6:e1001027)). To investigate fH binding beyond that to the fHbp ligand, fH binding was determined using an syngeneic mutant of group B strain H44 / 76 in which the gene encoding fHbp was inactivated (fHbp KO strain). Another mutant in which both the fHbp and NspA genes were inactivated served as a control to assess the potential role of NspA.

[0466] Flow cytometry results showed that the two mutant strains and the parental strain exhibited similar binding to the control mouse anti-PorA P1.7 mAb. Figure 40A As expected, the fHbp KO mutant bound to fH (100 μg / ml) less than the WT strain (in...). Figure 40B Compare the black and gray lines. In the absence of fHbp and NspA expression (dashed lines), fH binding was not different from the negative control WT bacteria (light gray bars) without the addition of fH. Similar results were obtained when 20% human serum with IgG removed was used as the source of fH. Figure 40C ).

[0467] Figure 40A-40CThe binding of fH to the group B H44 / 76 mutant results in the genetic inactivation of fHbp expression in the mutant, or the genetic inactivation of both fHbp and NspA expression. Figure 40A Anti-PorA mAb (P1.7, 20 μg / ml). Black line, WT; gray line, fHbp KO; dashed line, combination of fHbp KO and NspA KO. Figure 40B Binding of purified human fH (100 μg / ml). Identification and... Figure 40A Consistent with the above. Figure 40C Binding of fH in human serum (20%, IgG removed). Identification consistent with Figure 40A. Results of two independent assays were replicated.

[0468] The bactericidal activity against fHbp was detected using the syngeneic NspA KO mutant. Figures 41A-41E The study aimed to determine whether the binding of fH to NspA (and the corresponding downregulation of complement activation) might lead to a higher concentration of anti-fHbp mAb required for lysis in the presence of fH. Chimeric JAR 3 or JAR 5, which inhibits the binding of fH to fHbp, showed significantly higher bactericidal activity against the NspA KO mutant than the control WT strain (see [link to JAR 5]). Figure 41A and 41B Conversely, the chimeric mAb502, which does not inhibit fH binding, showed no bactericidal activity against any of the strains. Figure 41C Two control mouse mAbs, anti-PorA and anti-capsule, showed similar bactericidal activity against WT and mutant NspA KO strains (see [link to relevant documentation]). Figure 41D and 41E ).

[0469] Figures 41A-41E The bactericidal activity of group B H44 / 76 mutant against fHbp mAb was detected, in which NspA expression was genetically inactivated. Bacterial survival was assessed after incubation at 37°C for 60 minutes using each mAb and 20% human serum (IgG removed) as complement sources. Hollow triangle: NspA KO mutant; Solid triangle: Control WT strain. Figure 41A .Chimera JAR 3. Figure 41B .Chimera JAR 5. Figure 41C .Chimeric mAb502. Figure 41D Control mouse anti-Por A mAb (P 1.7). Figure 41E The control mouse mAb, SEAM 12, was reactive to the group B capsule. Results are presented from two experiments using three independent mAb dilutions. The results showed significant differences in survival between the WT and NspA KO strains after co-incubation with the mAb dilutions (*P≤0.02; **P<0.001).

[0470] The binding of fH to fHbp is crucial for the antifHbp mAb bactericidal activity. As shown above, using the NspA KO mutant of group B strain H44 / 76, data showed that fH binding to NspA was absent. In this case, the antifHbp mAb (JAR 3 or JAR 5) inhibiting fH binding exhibited higher bactericidal activity than when tested with wild-type strains containing NspA. A contrasting experiment was also conducted: anti-NspA mAb AL12 (Moe et al., Infect. Immun. (2002) 70:6021) was tested using the fHbp knockout mutant (Senegal 1 / 99) targeting group A strains. Figure 42 As shown, the fHbp KO mutant exhibited 50-fold higher bactericidal susceptibility to NspA mAb compared to the WT strain. Conversely, the control mAb against PorA P1.9 did not significantly enhance the bactericidal susceptibility of the fHbp KO mutant. The bactericidal activity of the mAbs was assessed using human complement (human serum with IgG removed).

[0471] Further data on the inhibitory effect of anti-fHbp antibodies on fH is significant for bactericidal activity. A prototype natural outer membrane vesicle (NOMV) vaccine was prepared from group B strain H44 / 76 overexpressing fHbp ID1. Antiserum from mice immunized with this vaccine showed bactericidal activity against eight of the nine tested African meningococcal isolates (Table 11). Conversely, all nine isolates were resistant to antiserum from mice immunized with the recombinant fHbp ID1 vaccine (bactericidal titer <1:10), and only one of the nine isolates was inactivated by control antiserum from mice immunized with the NOMV vaccine prepared using the fHbp KO mutant (X5, titer 1:36). Mixing the NOMV fHbp KO antiserum with antiserum against the recombinant fHbp ID1 vaccine did not increase bactericidal activity against any of the tested strains (Table 11). Therefore, the anti-fHbp antibodies induced by the NOMV vaccine overexpressing fHbp appear to result in bactericidal activity against isolates carrying fHbp sequence variants that do not match fHbp ID 1 in the NOMV vaccine. There is also no evidence that antibodies against fHbp share common bactericidal activity with antibodies against other antigens present in the NOMV vaccine.

[0472] Table 11. Bactericidal activity of serum from mice immunized with a natural outer membrane vesicle vaccine prepared by group B strain H44 / 76 overexpressing fHbp ID1.

[0473]

[0474] The mutant NOMV vaccine induced extensive serum cross-reactive anti-fHbp bactericidal activity, involving a higher anti-fHbp antibody response and a blocking effect on the binding of fH to fHbp compared to the recombinant fHbp vaccine. ELISA results showed that mice immunized with the NOMV vaccine prepared from the fHbp ID1 overexpressing mutant had higher serum anti-fHbp ID1 antibody concentrations (geometric means 2203 and 746 U / ml, respectively, P < 0.02) compared to mice immunized with the recombinant fHbp ID1 vaccine. Figure 43 (See Figure A). ELISA results showed that serum from mice immunized with the mutant NOMV vaccine also exhibited a higher inhibitory effect on the binding of fH to fHbp ID4, where fHbp ID4 is a sequence variant expressed by group A strain (…). Figure 43 (See Figure B, all tested dilutions showed P < 0.05). In the mutant NOMV vaccine group, the increased fH inhibition was not solely due to elevated serum anti-fHbp concentrations, as the mean concentration of anti-fHbp antibodies required for fH inhibition in this group was nearly 4 times lower than that in the recombinant fHbp vaccine ID 1 group (geometric means 1.17 and 4.04 U / ml, respectively, P < 0.05). Figure 43 (Small image C).

[0475] Figure 43Figure A. Anti-fHbp antibody response to vaccination was measured by ELISA (Figure A), and the inhibitory effect of serum anti-fHbp antibodies on the binding of fH to fHbp was measured by ELISA (Figures B and C, also by ELISA). Mice were immunized with a recombinant fHbp ID1 vaccine (solid triangle), or a NOMV vaccine prepared from a group B strain H44 / 76 mutant overexpressing fHbp ID1 (hollow circle) or fHbp knockout (solid circle). For the recombinant fHbp vaccine and the fHbp ID1 overexpressing NOMV vaccine, each symbol (Figures A and C) represents the result of one mouse (10 mice per vaccine group). For the NOMV fHbp KO vaccine group, each symbol represents the result of a test pool serum from 3 to 4 animals. A) Anti-fHbp ID1 antibody concentrations are expressed in arbitrary units per ml. The NOMV OE vaccine group had a higher geometric mean concentration (horizontal line) compared to mice immunized with the recombinant fHbp vaccine (p = 0.02). B) Inhibition of binding of fH to fHbp ID 4, which is heterologous to fHbp ID 1 in the vaccine. Among all dilutions, the mean inhibitory activity of the NOMV vaccine group (hollow circle) prepared from the fHbp overexpression mutant was higher than that of the recombinant fHbp vaccine group (solid triangle; p < 0.05). C) Concentration of serum anti-fHbp ID 1 antibody achieving 50% inhibition of binding of fH to fHbp ID 4 (which has 96% amino acid identity with ID 1). The geometric mean concentration of anti-fHbp antibody achieving 50% inhibition of fH binding in the NOMV OE fHbp group (hollow circle) was lower than that in the recombinant fHbp vaccine group (solid triangle, p < 0.05).

[0476] The present invention has been described through the specific embodiments described above. However, those skilled in the art should understand that various changes and equivalent substitutions can be made to the present invention without departing from its spirit and scope. Furthermore, various changes can be made to the purpose, spirit, and scope of the present invention to adapt to specific environments, materials, material compositions, processes, and process steps. All such changes should be considered to fall within the scope of the appended claims. sequence list <110> Children's Hospital & Research Center Atoakland <120> Modified Factor H Binding Protein (FHBP) and its Application <130> CHOR-057WO <150> US 61 / 440,227 <151> 2011-02-07 <150> US 61 / 423,757 <151> 2010-12-16 <150> US 61 / 381,025 <151> 2010-09-08 <150> US 61 / 334,542 <151> 2010-05-13 <150> US 61 / 319,181 <151> 2010-03-30 <160> 31 <170> FastSEQ version 4.0 for Windows operating systems <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> 262 <212> PRT <213> Neisseria meningitidis <400> 3 Cys Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Val Ala 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 Pro Gln Asn 35 40 45 Gly Thr Leu Thr Leu Ser Ala Gln Gly Ala Glu Lys Thr Phe Lys Ala 50 55 60 Gly Asp Lys Asp Asn Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp Lys 65 70 75 80 Ile Ser Arg Phe Asp Phe Val Gln Lys Ile Glu Val Asp Gly Gln Thr 85 90 95 Ile Thr Leu Ala Ser Gly Glu Phe Gln Ile Tyr Lys Gln Asn His Ser 100 105 110 Ala Val Val Ala Leu Gln Ile Glu Lys Ile Asn Asn Pro Asp Lys Thr 115 120 125 Asp Ser Leu Ile Asn Gln Arg Ser Phe Leu Val Ser Gly Leu Gly Gly 130 135 140 Glu His Thr Ala Phe Asn Gln Leu Pro Gly Gly Lys Ala Glu Tyr His 145 150 155 160 Gly Lys Ala Phe Ser Ser Asp Asp Pro Asn Gly Arg Leu His Tyr Ser 165 170 175 Ile Asp Phe Thr Lys Lys Gln Gly Tyr Gly Arg Ile Glu His Leu Lys 180 185 190 Thr Leu Glu Gln Asn Val Glu Leu Ala Ala Ala Glu Leu Lys Ala Asp 195 200 205 Glu Lys Ser His Ala Val Ile Leu Gly Asp Thr Arg Tyr Gly Ser Glu 210 215 220 Glu Lys Gly Thr Tyr His Leu Ala Leu Phe Gly Asp Arg Ala Gln Glu 225 230 235 240 Ile Ala Gly Ser Ala Thr Val Lys Ile Gly Glu Lys Val His Glu Ile 245 250 255 Gly Ile Ala Gly Lys Gln 260 <210> 4 <211> 254 <212> PRT <213> Neisseria meningitidis <400> 4 Cys Ser Ser Gly Gly Gly Gly Val Ala Ala Asp Ile Gly Ala Arg 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 Asp Gly Lys Ala Glu Tyr His Gly Lys Ala Phe Ser Ser Asp Asp 145 150 155 160 Ala Gly Gly Lys Leu Thr Tyr Thr Ile Asp Phe Ala Ala Lys Gln Gly 165 170 175 His Gly Lys Ile Glu His Leu Lys Thr Pro Glu Gln Asn Val Glu Leu 180 185 190 Ala Ala Ala Glu Leu Lys Ala Asp Glu Lys Ser His Ala Val Ile Leu 195 200 205 Gly Asp Thr Arg Tyr Gly Ser 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 Gly Glu Lys Val His Glu Ile Gly Ile Ala Gly Lys Gln 245 250 <210> 5 <211> 263 <212> PRT <213> Neisseria meningitidis <400> 5 Cys Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Val Ala Ala Asp 1 5 10 15 Ile Gly Ala 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 Arg Thr Phe Lys Ala 50 55 60 Gly Asp Lys Asp Asn Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp Lys 65 70 75 80 Ile Ser Arg Phe Asp Phe Ile Arg Gln Ile Glu Val Asp Gly Gln Leu 85 90 95 Ile Thr Leu Glu Ser Gly Glu Phe Gln Val Tyr Lys Gln Ser His Ser 100 105 110 Ala Leu Thr Ala Leu Gln Thr Glu Gln Val Gln Asp Ser Glu His Ser 115 120 125 Gly Lys Met Val Ala Lys Arg Gln Phe Arg Ile Gly Asp Ile Val Gly 130 135 140 Glu His Thr Ser Phe Gly Lys Leu Pro Lys Asp Val Met Ala Thr Tyr 145 150 155 160 Arg Gly Thr Ala Phe Gly Ser Asp Asp Ala Gly Gly Lys Leu Thr Tyr 165 170 175 Thr Ile Asp Phe Ala Ala Lys Gln Gly His Gly Lys Ile Glu His Leu 180 185 190 Lys Ser Pro Glu Leu Asn Val Asp Leu Ala Ala Ala Asp Ile Lys Pro 195 200 205 Asp Glu Lys His His Ala Val Ile Ser Gly Ser Val Leu Tyr Asn Gln 210 215 220 Ala Glu Lys Gly Ser Tyr Ser Leu Gly Ile Phe Gly Gly Gln Ala Gln 225 230 235 240 Glu Val Ala Gly Ser Ala Glu Val Glu Thr Ala Asn Gly Ile Arg His 245 250 255 Ile Gly Leu Ala Ala Lys Gln 260 <210> 6 <211> 255 <212> PRT <213> Neisseria meningitidis <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 Asn 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 Leu Gln Thr Glu 100 105 110 Gln Val Gln Asp Ser Glu His Ser Arg 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 Lys Gly Asp Ser 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 Tyr Gly Lys Ile Glu His Leu Lys Ser Pro Glu Leu Asn Val Asp 180 185 190 Leu Ala Ala Ala Tyr Ile Lys Pro Asp Glu Lys His His Ala Val Ile 195 200 205 Ser Gly Ser Val Leu Tyr Asn Gln Asp Glu Lys Gly Ser Tyr Ser Leu 210 215 220 Gly Ile Phe Gly Gly Gln Ala Gln Glu Val Ala Gly Ser Ala Glu Val 225 230 235 240 Lys Thr Ala Asn Gly Ile Arg His Ile Gly Leu Ala Ala Lys Gln 245 250 255 <210> 7 <211> 255 <212> PRT <213> Neisseria meningitidis <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 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 Val Tyr Lys Gln Ser His Ser Ala Leu Thr Ala Leu Gln Thr Glu 100 105 110 Gln Glu Gln Asp Pro Glu His Ser Gly Lys Met Val Ala Lys Arg Arg 115 120 125 Phe Lys Ile Gly Asp Ile Ala Gly Glu His Thr Ser Phe Asp Lys Leu 130 135 140 Pro Lys Asp Val Met 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 His Gly Lys Ile Glu His Leu Lys Ser Pro Glu Leu Asn Val Glu 180 185 190 Leu Ala Thr Ala Tyr Ile Lys Pro Asp Glu Lys His His Ala Val Ile 195 200 205 Ser Gly Ser Val Leu Tyr Asn Gln Asp Glu Lys Gly Ser Tyr Ser Leu 210 215 220 Gly Ile Phe Gly Gly Gln Ala Gln Glu Val Ala Gly Ser Ala Glu Val 225 230 235 240 Glu Thr Ala Asn Gly Ile His His Ile Gly Leu Ala Ala Lys Gln 245 250 255 <210> 8 <211> 254 <212> PRT <213> Artificial sequence <220> <223> fHbp ID 1 and ID 77 chimera <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 Ala Phe Asn Gln Leu 130 135 140 Pro Asp Gly Lys Ala Glu Tyr His Gly Lys Ala Phe Ser Ser Asp Asp 145 150 155 160 Ala Gly Gly Lys Leu Thr Tyr Thr Ile Asp Phe Ala Ala Lys Gln Gly 165 170 175 His Gly Lys Ile Glu His Leu Lys Thr Pro Glu Gln Asn Val Glu Leu 180 185 190 Ala Ala Ala Glu Leu Lys Ala Asp Glu Lys Ser His Ala Val Ile Leu 195 200 205 Gly Asp Thr Arg Tyr Gly Ser 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 Gly Glu Lys Val His Glu Ile Gly Ile Ala Gly Lys Gln 245 250 <210> 9 <211> 1231 <212> PRT <213> Homo sapiens <400> 9 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 Met 1010 1015 1020 Asp Gly Ala Ser Asn Val Thr Cys Ile Asn Ser Arg Trp Thr Gly Arg 1025 1030 1035 1040 Pro Thr Cys Arg Asp Thr Ser Cys Val Asn Pro Pro Thr Val Gln Asn 1045 1050 1055 Ala Tyr Ile Val Ser Arg Gln Met Ser Lys Tyr Pro Ser Gly Glu Arg 1060 1065 1070 Val Arg Tyr Gln Cys Arg Ser Pro Tyr Glu Met Phe Gly Asp Glu Glu 1075 1080 1085 Val Met Cys Leu Asn Gly Asn Trp Thr Glu Pro Pro Gln Cys Lys Asp 1090 1095 1100 Ser Thr Gly Lys Cys Gly Pro Pro Pro Pro Ile Asp Asn Gly Asp Ile 1105 1110 1115 1120 Thr Ser Phe Pro Leu Ser Val Tyr Ala Pro Ala Ser Ser Val Glu Tyr 1125 1130 1135 Gln Cys Gln Asn Leu Tyr Gln Leu Glu Gly Asn Lys Arg Ile Thr Cys 1140 1145 1150 Arg Asn Gly Gln Trp Ser Glu Pro Pro Lys Cys Leu His Pro Cys Val 1155 1160 1165 Ile Ser Arg Glu Ile Met Glu Asn Tyr Asn Ile Ala Leu Arg Trp Thr 1170 1175 1180 Ala Lys Gln Lys Leu Tyr Ser Arg Thr Gly Glu Ser Val Glu Phe Val 1185 1190 1195 1200 Cys Lys Arg Gly Tyr Arg Leu Ser Ser Arg Ser His Thr Leu Arg Thr 1205 1210 1215 Thr Cys Trp Asp Gly Lys Leu Glu Tyr Pro Thr Cys Ala Lys Arg 1220 1225 1230 <210> 10 <211> 50 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 10 Thr Ala Pro Leu Asp His Lys Asp Lys Gly Leu Gln Ser Leu Thr Leu 1 5 10 15 Asp Gln Ser Val Arg Lys Asn Glu Lys Leu Lys Leu Ala Ala Gln Gly 20 25 30 Ala Glu Lys Thr Tyr Gly Asn Gly Asp Ser Leu Asn Thr Gly Lys Leu 35 40 45 Lys Asn 50 <210> 11 <211> 50 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 11 Thr Ala Pro Leu Asp His Lys Asp Lys Ser Leu Gln Ser Leu Thr Leu 1 5 10 15 Asp Gln Ser Val Arg Lys Asn Glu Lys Leu Lys Leu Ala Ala Gln Gly 20 25 30 Ala Glu Lys Thr Tyr Gly Asn Gly Asp Ser Leu Asn Thr Gly Lys Leu 35 40 45 Lys Asn 50 <210> 12 <211> 53 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 12 Thr Ala Pro Leu Asp His Lys Asp Lys Gly Leu Lys Ser Leu Thr Leu 1 5 10 15 Glu Asp Ser Ile Ser Gln Asn Gly Thr Leu Thr Leu Ser Ala Gln Gly 20 25 30 Ala Glu Arg Thr Phe Lys Ala Gly Asp Lys Asp Asn Ser Leu Asn Thr 35 40 45 Gly Lys Leu Lys Asn 50 <210> 13 <211> 50 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 13 Thr Ala Pro Leu Asp His Lys Asp Lys Gly Leu Lys Ser Leu Thr Leu 1 5 10 15 Glu Asp Ser Ile Ser Gln Asn Gly Thr Leu Thr Leu Ser Ala Gln Gly 20 25 30 Ala Glu Lys Thr Tyr Gly Asn Gly Asp Ser Leu Asn Thr Gly Lys Leu 35 40 45 Lys Asn 50 <210> 14 <211> 53 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 14 Thr Ala Pro Leu Asp His Lys Asp Lys Gly Leu Lys Ser Leu Thr Leu 1 5 10 15 Glu Asp Ser Ile Pro Gln Asn Gly Thr Leu Thr Leu Ser Ala Gln Gly 20 25 30 Ala Glu Lys Thr Phe Lys Ala Gly Asp Lys Asp Asn Ser Leu Asn Thr 35 40 45 Gly Lys Leu Lys Asn 50 <210> 15 <211> 174 <212> PRT <213> Neisseria meningitidis <400> 15 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> 16 <211> 19 <212> PRT <213> artificial sequence <220> <223> Synthetic peptides <400> 16 Met Asn Arg Thr Ala Phe Cys Cys Leu Ser Leu Thr Thr Ala Leu Ile 1 5 10 15 Leu Thr Ala <210> 17 <211> 66 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 17 Val Ala Ala Asp Ile Gly Ala Gly Leu Ala Asp Ala Leu Thr Ala Pro 1 5 10 15 Leu Asp His Lys Asp Lys Ser Leu Gln Ser Leu Thr Leu Asp Gln Ser 20 25 30 Val Arg Lys Asn Glu Lys Leu Lys Leu Ala Ala Gln Gly Ala Glu Lys 35 40 45 Thr Tyr Gly Asn Gly Asp Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp 50 55 60 Lys Val 65 <210> 18 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 18 Phe Leu Leu Ala Val 1 5 <210> 19 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 19 Gly Ser Gly Gly Ser 1 5 <210> 20 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 20 Gly Gly Gly Ser 1 <210> twenty one <211> 4 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> twenty one Gly Gly Ser Gly 1 <210> twenty two <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> twenty two Gly Gly Ser Gly Gly 1 5 <210> twenty three <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> twenty three Gly Ser Gly Ser Gly 1 5 <210> twenty four <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> twenty four Gly Ser Gly Gly Gly 1 5 <210> 25 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 25 Gly Gly Gly Ser Gly 1 5 <210> 26 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 26 Gly Ser Ser Ser Gly 1 5 <210> 27 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 27 Gly Glu His Thr 1 <210> 28 <211> 66 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 28 Val Ala Ala Asp Ile Gly Ala Gly Leu Ala Asp Ala Leu Thr Ala Pro 1 5 10 15 Leu Asp His Lys Asp Lys Gly Leu Gln Ser Leu Thr Leu Asp Gln Ser 20 25 30 Val Arg Lys Asn Glu Lys Leu Lys Leu Ala Ala Gln Gly Ala Glu Lys 35 40 45 Thr Tyr Gly Asn Gly Asp Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp 50 55 60 Lys Val 65 <210> 29 <211> 66 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 29 Val Ala Ala Asp Ile Gly Ala Arg Leu Ala Asp Ala Leu Thr Ala Pro 1 5 10 15 Leu Asp His Lys Asp Lys Ser Leu Gln Ser Leu Thr Leu Asp Gln Ser 20 25 30 Val Arg Lys Asn Glu Lys Leu Lys Leu Ala Ala Gln Gly Ala Glu Lys 35 40 45 Thr Tyr Gly Asn Gly Asp Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp 50 55 60 Lys Val 65 <210> 30 <211> 60 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 30 Val Tyr Lys Gln Ser His Ser Ala Leu Thr Ala Phe Gln Thr Glu Gln 1 5 10 15 Ile Gln Asp Ser Glu His Ser Gly Lys Met Val Ala Lys Arg Gln Phe 20 25 30 Arg Ile Gly Asp Ile Ala Gly Glu His Thr Ser Phe Asp Lys Leu Pro 35 40 45 Glu Gly Gly Arg Ala Thr Tyr Arg Gly Thr Ala Phe 50 55 60 <210> 31 <211> 60 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 31 Val Tyr Lys Gln Ser His Ser Ala Leu Thr Ala Leu Gln Thr Glu Gln 1 5 10 15 Val Gln Asp Ser Glu His Ser Gly Lys Met Val Ala Lys Arg Gln Phe 20 25 30 Arg Ile Gly Asp Ile Val Gly Glu His Thr Ser Phe Gly Lys Leu Pro 35 40 45 Lys Asp Val Met Ala Thr Tyr Arg Gly Thr Ala Phe 50 55 60

Claims

1. A variant of factor H binding protein (fHbp), said variant fHbp differing from fHbp of SEQ ID NO:2 only at position 210, wherein, The amino acid at position 210 of the variant fHbp is alanine.

2. The variant fHbp of claim 1, wherein the affinity of the variant fHbp for binding human fH is 85% to 75%, 75% to 65%, 65% to 55%, 55% to 45%, 45% to 35%, 35% to 25%, 25% to 15%, 15% to 10%, 10% to 5%, 5% to 2%, 2% to 1%, or 1% to 0.1% of the affinity of fHbp ID22 for binding human fH.

3. The variant fHbp of claim 1, wherein the variant fHbp induces levels of *Neisseria meningitidis* in mammals immunized with the variant fHbp. Neisseria meningitidis The level of serum bactericidal antibodies was comparable to or higher than the level of serum bactericidal antibodies against Neisseria meningitidis induced in mammals immunized with fHbp ID22.

4. A composition comprising the variant fHbp according to any one of claims 1-3 and a pharmaceutically acceptable excipient.

5. The composition according to claim 4, wherein the variant fHbp is in a vesicle formulation prepared from a strain of Neisseria meningitidis.

6. An immunogenic composition comprising: a) Natural outer membrane vesicles obtained from genetically modified Neisseria meningitidis host cells, wherein the genetically modified Neisseria meningitidis host cells are genetically modified with nucleic acid encoding a variant fHbp according to any one of claims 1-3, such that the variant fHbp encoded is produced by the genetically modified host cells, wherein the vesicles comprise the variant fHbp encoded; and b) Pharmaceutically acceptable excipients.

7. The composition according to any one of claims 4-6, for use in inducing an antibody response against Neisseria meningitidis in a mammal, wherein the mammal is a human.

8. The composition according to any one of claims 4-6, used in the method of claim 7, wherein the antibody response comprises generating a bactericidal antibody against Neisseria meningitidis.

9. A nucleic acid encoding a variant fHbp of any one of claims 1-3.

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

11. A genetically modified Neisseria meningitidis host cell, comprising: The nucleic acid as described in claim 9; or The recombinant expression vector according to claim 10.

12. Use of the composition according to any one of claims 4-6 in the preparation of a medicament for inducing an antibody response against Neisseria meningitidis in a mammal, wherein the mammal is a human.

13. The use according to claim 12, wherein the antibody response comprises generating a bactericidal antibody against Neisseria meningitidis.

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