WO2009114485A2 - Chimeric factor h binding proteins (fhbp) containing a heterologous b domain and methods of use - Google Patents

Chimeric factor h binding proteins (fhbp) containing a heterologous b domain and methods of use Download PDF

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WO2009114485A2
WO2009114485A2 PCT/US2009/036577 US2009036577W WO2009114485A2 WO 2009114485 A2 WO2009114485 A2 WO 2009114485A2 US 2009036577 W US2009036577 W US 2009036577W WO 2009114485 A2 WO2009114485 A2 WO 2009114485A2
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fhbp
domain
amino acid
chimeric
acid sequence
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PCT/US2009/036577
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French (fr)
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WO2009114485A3 (en
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Dan Granoff
Peter Beernink
Jo Anne Welsch
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Children's Hospital & Research Center At Oakland
Novartis Vaccines And Diagnostics, Srl.
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Priority to AU2009223613A priority Critical patent/AU2009223613B2/en
Priority to CA2717870A priority patent/CA2717870A1/en
Priority to EP09720813.6A priority patent/EP2265640B1/en
Priority to US12/921,123 priority patent/US9511131B2/en
Priority to ES09720813.6T priority patent/ES2557282T3/en
Publication of WO2009114485A2 publication Critical patent/WO2009114485A2/en
Publication of WO2009114485A3 publication Critical patent/WO2009114485A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/095—Neisseria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1217—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Neisseriaceae (F)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/22—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Neisseriaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • This invention relates to vaccines for diseases caused by Neisseria meningitidis.
  • Neisseria meningitidis is a Gram-negative bacterium which colonizes the human upper respiratory tract and is responsible for worldwide sporadic and cyclical epidemic outbreaks of, most notably, meningitis and sepsis. The attack and morbidity rates are highest in children under 2 years of age. Like other Gram-negative bacteria, Neisseria meningitidis typically possess a cytoplasmic membrane, a peptidoglycan layer, an outer membrane which together with the capsular polysaccharide constitute the bacterial wall, and pili, which project into the outside environment. Encapsulated strains of Neisseria meningitidis are a major cause of bacterial meningitis and septicemia in children and young adults. The prevalence and economic importance of invasive Neisseria meningitidis infections have driven the search for effective vaccines that can confer immunity across different strains, and particularly across genetically diverse group B strains with different serotypes or serosubtypes.
  • fHBP Factor H Binding Protein
  • N. meningitidis protein which is expressed in the bacterium as a surface-exposed lipoprotein. Based on sequence analysis of 71 N. meningitidis strains representative of its genetic and geographic diversity, N.
  • variant 1 meningitidis strains have been sub-divided into three fHBP variant groups (referred to as variant 1 (v.l), variant 2 (v.2), and variant 3 (v.3)) based on amino acid sequence variability and immunologic cross-reactivity (Masignani et al. J Exp Med 2003; 197:789-99).
  • Other workers (Fletcher et al, 2004) have subdivided the protein into two sub-families designated A (which includes v.2 and v.3 of Masignani) and B (v.l).
  • Variant 1 strains account for about 60% of disease-producing group B isolates (Masignani et al. 2003, supra).
  • each variant group there is on the order of about 92% or greater conservation of amino acid sequence. Specifically, conservation within each variant group ranges between 89 and 100%, while between the variant groups (e.g., between v.l and v.2) the conservation can be as low as 59%.
  • the protein is expressed by all known strains of N. meningitidis. [0006] Mice immunized with recombinant fHBP developed high serum bactericidal antibody responses against strains expressing fHBP proteins of the homologous variant group (Masignani et al. 2003, supra; Welsch et al. 2004, J Immunol. 172(9):5606-15.).
  • antiserum prepared against fHBP v.l confers protection against N. meningitidis strains expressing fHBP v.l, but not against strains expressing fHBP v.2 or v.3.
  • antiserum prepared against fHBP v.2 protects against strains expressing v.2 (or v.3) but not v.l (Masignani et al. J Exp Med 2003, 197:789-99; Beernink et al. J Infect Dis 2007; 195: 1472- 9).
  • Chimeric proteins have been used for vaccine development in a variety of ways.
  • a first strategy employs a genetic or chemical linkage of an antigen to a known, but unrelated, immunogenic protein, such as the diphtheria, tetanus or pertussis toxoid proteins, or the cholera toxin B (CTB) domain, in order to enhance the magnitude of the antibody responses to the antigen of interest.
  • CTB cholera toxin B
  • a second strategy uses a genetic fusion of two antigens from the same organism, to enhance cross-protection against strains with antigenic diversity (Giuliani et al. Infect Immun 2005 73:1151-60).
  • An example is the multivalent group B meningococcal recombinant protein vaccine, which contains a mixture of two fusion proteins: a first fusion protein of a G ⁇ A2091 protein and a GNAl 870 (or "fHBP") protein, and a second fusion protein of a GNA2132 protein and a GNA1030 protein (Giuliani et al. Proc Natl Acad Sci U S A 2006, 103:10834-9).
  • a third strategy has been to construct a fusion of different serologic variants ("serovars") of one antigen to induce cross-
  • An example is a tetravalent OspC chimeric Lyme disease vaccine, which induced bactericidal antibody responses against spirochete strains expressing each of the OspC types that were incorporated into the construct (Earnhart et al. Vaccine 2007;25:466-80).
  • the vaccines were composed of repeats of an individual domain with antigenic variability.
  • the respective variants of the domain were expressed in tandem in one protein (i.e., the same domain from different strains, A 1 -A 2 -A 3 -A 4 , etc).
  • these recombinant tandem proteins can be convenient for manufacturing and quality control. However they also can be very large and subject to improper folding or degradation.
  • One approach to avoiding the problem of large tandem fusion proteins is to design a single polypeptide that is composed of different domains of two antigenic variants e.g., by "swapping" different individual domains of an antigen, or even smaller regions such as individual epitopes from two different proteins, to form a chimeric protein that expresses antigenically unrelated epitopes specific for more than one strain (i.e., different domains from two different strains, A 1 -B 2 or A 2 -B 1 , etc.).
  • a chimeric protein containing the B domain from a variant 3 strain (B 3 ) fused with the C domain of a variant 1 strain (C 1 ) failed to elicit protective bactericidal responses against strains with either v.l or v.2 fHBP.
  • Vaccines that exploit the ability of fHBP to elicit bactericidal antibody responses and that can elicit such antibodies that are effective against strains expressing different fHBP variants remain of interest.
  • Figure 1 is a collection of results of Western blot analysis illustrating the amino acid residues involved in binding of monoclonal antibodies (mAbs) JAR 3 and JAR 5 to factor H binding protein (fHBP).
  • Panel A JAR 5; lane 1, molecular mass standard; lane 2, pET21b; lane 3, pET21-fHBP(MC58 wildtype); lane 4, pET21-fHBP(MC58)G121R; lane 5, pET21-fHBP(M6190 wildtype)R121; lane 6, pET21-fHBP(M6190)R121G.
  • Panel B JAR 3.
  • C Penta-His mAb. Panels B and C have the same lane assignments as panel A.
  • FIG. 2 is a set of graphs illustrating that binding of JAR 3 and JAR 5 mAbs to fHBP is competitive. Percent competitive inhibition of binding of anti-fHBP mAbs to fHBP by a second antibody as measured by ELISA. Each panel includes: rabbit polyclonal anti-fHBP antiserum; rabbit pre-immune serum; and a negative control mAb specific for an irrelevant capsular antigen (JW-C2, -A2 or -Al). Panel A, Inhibition of binding of JAR 3 by JAR 4 or JAR 5. Panel B, Inhibition of binding of JAR 5 by JAR 3 or JAR 4. Panel C, Inhibition of binding of JAR 4 by JAR 3 or JAR 5.
  • Figure 3 is a schematic illustrating positions of residues associated with the epitopes of the nine anti-fHBP mAbs ("JAR" mAbs) in the structural model based on previously reported NMR data (Cantini et al. "Solution structure of the immunodominant domain of protective antigen GNA1870 of Neisseria meningitidis .” J Biol Chem 2006; 281:7220-7). Coordinates from the solution structure of the B and C domains of fHBP v.l from strain MC58 were used to construct the model.
  • JAR anti-fHBP mAbs
  • variant 2 (v.2) fHBP protein (from strain 8047) and variant 3 (v.3) fHBP (from strain M1239) differ by -1 and +7 amino acid residues, respectively, from that of MC58
  • the numbering used to refer to residues for v.2 and v.3 fHBP proteins differs from numbering based on the actual amino acid sequences of these proteins.
  • reference to a leucine residue (L) at position 166 of the v.2 or v.3 fHBP sequence in Figure 3 refers to the residue at position 165 of the v.2 protein and at position 173 in the v.3 protein.
  • Figure 4 for alignment.
  • the residue shown for mAb 502 is from a previously reported study (Giuliani et al., 2005 Infect Immun 73:1151-60). The numbering is based on amino acid sequence of MC58 v.l fHBP lacking the signal sequence (Masignani et al., 2003 J Exp Med 197:789-99).
  • Figure 4 is a schematic providing an alignment of wild-type and chimeric fHBP amino acid sequences (alignment performed using ClustalW).
  • the deduced amino acid sequences of fHBP v.l from strain MC58 (bottom) and v.2 strain 8047 (top) are shown, along with the two chimeric sequences (middle, Chimera I and Chimera II). Numbering for all four proteins is based on the native, mature v.l protein from MC58 (i.e., without the signal sequence).
  • the recombinant fHBP protein as expressed in E. coli lacks both the signal sequence and seven presumably flexible residues (CSSGGGG).
  • Chimera I and Chimera II have identical amino acid sequences. The exception is at residue 174 where alanine in Chimera I has been replaced by lysine in Chimera II. The position of the A174K substitution in Chimera II is shown in bold. Sequence alignment was performed with ClustalW.
  • Figure 5 is a schematic representation of chimeric fHBPs.
  • the N-terminal portion of the B domain from the v.1 fHBP is on the right, and the C-terminal portion of the B domain encompassing the ⁇ -helix of the v.2 protein together with the C domain of the v. 2 fHBP is on the left.
  • the junction point for these chimeras, exemplified by G136 is indicated with an arrow and accompanying text.
  • Chimera I contains the JAR 11 epitope, including residue A174 (Panel A), which is expressed on the C domains of a subset of strains expressing fHBP v.2 or v.3.
  • Chimera II contains the JAR 32 /JAR 35 epitopes, including residue K174, which are expressed on the C domains of subsets of strains expressing fHBP v.2 or v.3. A domains are not shown in these representations.
  • the model was constructed based on the NMR structure of Cantini et al. (2006) J Biol Chem 281:7220-7.
  • Figure 6 provides the results of SDS-PAGE analysis of purified wild-type and mutant fHBPs. Proteins were expressed from pET21-based plasmids in E. coli BL21(DE3) as C-terminal hexa-histidine fusions and purified by metal chelate chromatography. Proteins were dialyzed against Ix PBS, 5% sucrose, 1 mM DTT and filter sterilized. Proteins (5 ⁇ g each) were separated on a 4-12% polyacrylamide gel and stained with Coomassie blue. Lane 1, mass standard; 2, fHBP v.l (MC58); 3, fHBP v.2 (8047); 4, fHBP Chimera I; 5, fHBP Chimera II.
  • Figure 7 is a set of graphs illustrating binding of individual anti-fHBP mAbs to recombinant proteins.
  • Panel A shows mAbs prepared against fHBP v.l;
  • Panel B fHBP v.2;
  • Panel C fHBP v.3.
  • the symbols represent different antigens on the plate: open squares, fHBP v.l; open circles, fHBP v.2; open triangles, Chimera I; asterisks, Chimera II.
  • Figure 8 A provides a table of strains used in the Examples, including those used to measure serum bactericidal antibody responses and description of the amino acid sequence identity compared with prototype fHBP v.l, v.2 and v.3 and JAR mAb binding of the respective fHBPs.
  • Figure 8B shows the amino acid identities of different domains of fHBP.
  • Comparisons are made for the A domain (residues 1-100), the B domain (residues 101-164) and the C domain (residues 165-255). Comparisons also are made for the B domain up to the junction point (101-135) and the B domain starting at the junction point (136-164). Numbering of amino acid residues is based on the mature protein (i.e. lacking the signal sequence) from strain MC58.
  • Figure 9 is a graph illustrating serum bactericidal antibody responses of mice immunized with chimeric recombinant proteins given with Freund's adjuvant as measured against N. meningitidis group B strains expressing fHBP in the antigenic v.l group. Strain H44/76 expresses fHBP v.1 identical to that of the fHBP v.1 control vaccine. The remaining strains express subvariants of fHBP v.l (See Table in Figure 8A, above). Values are presented as 1/GMT (Reciprocal (or inverse) geometric mean titer) with a 95% confidence interval.
  • 1/GMT Reciprocal (or inverse) geometric mean titer
  • Figure 10 is a graph illustrating serum bactericidal antibody responses of mice immunized with chimeric recombinant proteins given with Freund's adjuvant as measured against N. meningitidis group B expressing fHBP in the v.2 or v.3 antigenic groups.
  • fHBP v.2 expresses fHBP v.2 identical to that of control rfHBP v.2 vaccine.
  • the remaining strains express subvariants of fHBP v.2 or v.3 (see Table in Figure 8A). Values are presented as 1/GMT with a 95% confidence interval.
  • the data are stratified based on strains reacting with JAR 11 (left panel) or JAR 32 (right panel).
  • the Chimera I and II vaccines are identical except that Chimera I has residue A174 and is JAR 11-positive and JAR 32-negative, whereas Chimera II has residue K174 and is JAR 11-negative and JAR 32-positive. See figure for bar symbols.
  • Figure 11 is a graph illustrating serum bactericidal antibody responses of mice immunized with chimeric recombinant proteins adsorbed to aluminum hydroxide as measured against N. meningitidis group B strains expressing fHBP in the antigenic v.l group. Strain H44/76 expresses fHBP v.l identical to that of the fHBP v.l control vaccine. The remaining strains express subvariants of fHBP v.l. Values are presented as 1/GMT (i.e., reciprocal (or inverse) geometric mean titer) with a 95% confidence interval. Bar symbols for each vaccine are as shown in Figure 10.
  • 1/GMT i.e., reciprocal (or inverse) geometric mean titer
  • Figure 12 is a graph illustrating serum bactericidal antibody responses of mice immunized with chimeric recombinant proteins adsorbed to aluminum hydroxide as measured against N. meningitidis group B expressing fHBP in the v.2 or v.3 antigenic groups.
  • Strain 8047 expresses fHBP v.2 identical to that of control rfHBP v.2 vaccine. The remaining strains express subvariants of fHBP v.2 or v.3 (see Table in Figure 8). Values are presented as 1/GMT with a 95% confidence interval. The data are stratified based on strains reacting with JAR 11 (left panel) or JAR 32 (right panel).
  • FIG. 13 is a schematic showing alignment of fHBP v.l amino acid sequences with natural polymorphisms in the ⁇ -terminal portion of the B domain.
  • the sequence shown also comprises a C-terminal portion of the fHbp ⁇ domain and a small ⁇ -terminal portion of the fHbpC domain as indicated above the alignment. The sequence conservation is shown below the alignment (code as in Figure 4).
  • strains MC48, M4105, 4243, NZ98/254 are positive for JAR3/JAR 5 reactivity; strains M6190 and 03S-0408 are negative for JAR 3/5 reactivity, and strains NM452 and CDC1573 have not been tested.
  • the residues G121 and K122, which are associated with JAR 3 and JAR 5 mAb epitopes, are shown in bold and underlined text. Note that although strain 03S-0408 has G121, it is negative for JAR 3/5 reactivity.
  • This strain has three amino acid differences between positions 101 and 146 compared with amino acids of MC58: L109, V114 and S122. Since both L109 and V114 are present in reactive sequences, e.g. 4243 and M4105, lack of reactivity of 03S-0408 is likely attributable to the presence of serine at position 122 instead of lysine and, therefore in addition to G121, K122 also is associated with JAR3/5 reactivity.
  • Figure 14 is a schematic showing alignment of fHBP v.2 amino acid sequences with natural polymorphisms in the carboxyl-terminal portion of the B domain and the C domain, or alternatively, the complete fHbpC domain based on the three-dimensional structural nomenclature. The sequence conservation is shown below the alignment (code as in legend to Figure 4).
  • the residues implicated in anti-fHBP mAb epitopes are designated with the number of the JAR mAb above the alignment: JAR 11 (alanine at residue position 174; A174); JAR 10 (lysine at residue position 180 and glutamate at position 192; K180 and E192); JAR 13 (serine residue at position 216; S216). Numbering in this figure is based on fHBP from strain MC58.
  • Figure 15 is a schematic illustrating additional exemplary chimeric vaccines
  • Chimera lib can be made by introducing the Kl 80R substitution into Chimera II.
  • Chimeras III and V can be made using portions of the A and B domains of strain NZ98/254 (subvariant v.1) with the distal portion of the B domain and C domain of v.2 strain 8047 (Chimera III) or of subvariant v.2 strain RM1090 (Chimera V).
  • Chimera IV uses the A and proximal B domains of MC58 with the distal B and C domains of RM 1090.
  • Figure 16 is a schematic showing an alignment of amino acid sequences of further exemplary chimeric fHBPs (Chimera III, IV and V) in the region of the crossover position, which is indicated by the box (residues GEHT). The residues, G121 and K122, implicated in the JAR 3 and JAR 5 epitopes are shown in bold and underlining.
  • Figure 17 provides a table summarizing cross-reactivity of the different JAR mAbs, their respective Ig isotypes and ability to inhibit binding of human fH.
  • Figure 18 is a table listing human complement- mediated bactericidal activity of each of the JAR mAbs when tested individually or in combination with a second anti- fHBP mAb.
  • Figure 19 is a series of graphs showing the ability of representative JAR mAbs prepared against fHBP v.2 or v.3 proteins to give concentration-dependent inhibition of binding of fH to rfHBP in an ELISA.
  • Panel A Inhibition of binding of fH to rfHBP v.2.
  • Panel B Inhibition of binding of fH to rfHBP v.3.
  • Respective v.2 and v.3 recombinant proteins are those encoded by the fHBP genes of strains 8047 and M1239.
  • Panel C Inhibition of binding of fH to rfHBP v.1.
  • Figure 20 is a table listing certain properties of respective pairs of JAR mAbs with or without synergistic complement-mediated bactericidal antibody, including the positions of amino acid residues involved in the epitopes, distances between them, inhibition of fH binding and isotype of each mAb.
  • Figure 21 provides the amino acid sequence of variant 1 (v.l) factor H binding protein (fHBP) of MC58, with the A, B and C domains indicated. Positions of the structural domains, fHbpN and fHbpC, are also shown.
  • Glutamine 101 (Q) and glycine 164 (G) indicated by upward arrows define the A/B and B/C domain borders, respectively, as defined by Giuliani et al., Infect. Immun, 2005 73:1151-60.
  • the upward arrow at glycine 136 designates the boundary between the fHbpN and the fHbpC domains, as defined by Cantini et al., J. Biol. Chem. 2009.
  • Figure 22 shows the amino- (N-) and carboxyl- (C-) terminal portions of the B and C domains, which are defined with respect to the conserved amino acid sequence of GEHT.
  • the amino acid sequences that can define a JAR 3/5 epitope are positioned N- terminal to the second alpha helix; the amino acid sequence that can define the JAR 11/32/35 epitopes are positioned C-terminal to the second alpha helix.
  • Alpha-helix (AH) 2 is indicated.
  • Figure 23 is a schematic showing Chimera I (MC58/8047) nucleotide and protein sequences. The sequence before the junction (cross-over) point is shown in lower case and the sequence following the junction point is shown in upper case.
  • FIG. 24 is a schematic showing Chimera II (MC58/8047 A 174K) nucleotide and protein sequences. The sequence before the junction (cross-over) point is shown in lower case and the sequence following the junction point is shown in upper case. Lines of fifty residues are shown. Only the Neisserial sequences are shown; E. coli expression constructs contained an N-terminal Methionine and C-terminal hexa-histidine tag (LEHHHHHH). The A174K substitution is shown in bold and underlined text.
  • Figure 25 is a schematic showing Chimera lib (MC58/8047 A174K/K180R) nucleotide and protein sequences. The sequence before the junction (cross-over) point is shown in lower case and the sequence following the junction point shown in upper case. Lines of fifty residues are shown. Only the Neisserial sequences are shown; E. coli expression constructs contain an N-terminal Methionine and C-terminal hexa-histidine tag (LEHHHHHH). The A174K and Kl 8OR substitutions are shown in bold text.
  • Figure 26 is a schematic showing Chimera III (NZ98254/8047) nucleotide and protein sequences.
  • FIG. 27 is a schematic showing Chimera IV (MC58/RM1090) nucleotide and protein sequences. The sequence before the junction (cross-over) point is shown in lower case and the sequence following the junction point is shown in upper case. Lines of fifty residues are shown. Only the Neisserial sequences are shown; E. coli expression constructs contain an N-terminal Methionine and C-terminal hexa-histidine tag (LEHHHHHH).
  • Figure 27 is a schematic showing Chimera IV (MC58/RM1090) nucleotide and protein sequences. The sequence before the junction (cross-over) point is shown in lower case and the sequence following the junction point is shown in upper case. Lines of fifty residues are shown. Only the Neisserial sequences are shown; E.
  • FIG. 28 is a schematic showing Chimera V (NZ98254/RM1090) nucleotide and protein sequences. The sequence before the junction (cross-over) point is shown in lower case and the sequence following the junction point is shown in upper case. Lines of fifty residues are shown. Only the Neisserial sequences are shown; E. coli expression constructs contain an N-terminal methionine and C-terminal hexa-histidine tag (LEHHHHHH).
  • Figure 29 provides a table showing positions of residues associated with JAR mAb binding.
  • the reactive residue in fHBP was from the strain used as the source for immunization.
  • the reactive strain is 8047, whose fHBP sequence is 99.6% identical to that from strain 2996.
  • the non-reactive residue is that present in the non- reactive strain. Loss of reactivity associated with a change from the reactive to the non- reactive residue is indicated as knock-out (KO) and the converse change is indicated as knock-in (KI).
  • Figure 30 provides an image of a Western blot indicating residues involved in the JAR 10 and JAR 33 epitopes.
  • E. coli lysates containing plasmids expressing the respective wild-type and mutant fHBPs were analyzed by Western blot with JAR 10 (Panel A), Penta-His mAb (Panel B), or JAR 33 (Panel C).
  • Figure 31 provides an image of a Western blot indicating a residue involved in the JAR 11, JAR 32 and JAR 35 epitopes.
  • E. coli lysates containing plasmids expressing the respective wild-type and mutant fHBPs were analyzed by Western blot with JAR 32 (Panel A), JAR 35 (Panel B), JAR 11 (Panel C) or Penta-His mAb (Panel D).
  • Figure 32 provides an image of a Western blot indicating residue involved in the JAR 13 epitope.
  • coli lysates containing plasmids expressing wild-type and mutant fHBPs lane 1, molecular weight marker; lane 2, pET21 (empty plasmid); lane 3, fHBP(8047)wt; lane 4, fHBP(8047)S216G; lane 5, fHBP(RM1090)wt; lane 6, fHBP(RM1090)G216S. Blots were probed with JAR 13 (Panel A) or Penta-His mAb (Panel B) and anti-mouse IgG-HRP secondary antibody.
  • Figure 33 provides an image of a Western blot of wildtype (WT) or Chimeric fHBP expressed in N. meningitidis.
  • Lane 1 H44/76 KO fHBP transformed with pCom-fHBP v.2 WT plasmid; 2, H44/76 KO fHbp transformed with pCom-Chimera I plasmid; 3, Kaleidoscope marker; 4, Magic Mark marker; 5, H44/76 (v.l) WT cells; 6, 8047 (v.2) WT cells; 7, 8047 KO fHBP cells; 8, recombinant (r) fHBP v.l protein (gene from strain MC58); 9, rfHBP v.2 protein (gene from strain 8047).
  • FIG. 34 shows ribbon diagrams of full length v.1 fHBPs.
  • Panel A, fHBP is partitioned into three domains indicated by various shades of gray.
  • the A domain and the ⁇ - terminal portion of the B domain are on the left and the boundary between the A and B domains is indicated by an arrow at lysine 100.
  • the C-terminal portion of the B domain together with the C domain is on the right, where the boundary between the two is designated by an arrow at glycine 164.
  • Panel B an alternative nomenclature describes the fHBP as having two structural domains.
  • the ⁇ -terminal domain containing a mix of ⁇ helices and ⁇ strands is named the fHbp ⁇ domain (left) and the C-terminal domain consisting of ⁇ strands is labeled as the fHbpC domain (right).
  • the fHbp ⁇ and the fHbpC are connected by a linker at or proximal to glycine 136.
  • the junction point relevant for the chimeric fHBP described herein is at or proximal to G136, indicated by an arrow in both panels.
  • the models shown in both panels are constructed based on the ⁇ MR structure of Cantini et al. J Biol Chem 2009.
  • the present disclosure provides chimeric fHBPs that can elicit antibodies that are bactericidal for different fHBP variant strains of N. meningitidis, and methods of use.
  • fHBP Fractor H Binding Protein
  • GNA1870, GNA 1870, ORF2086, LP2086 (lipoprotein 2086), and "741" refers to a polypeptide of N. meningitidis that is a lipoprotein presented on the surface of the bacterium.
  • N. meningitidis strains have been sub-divided into three fHBP variant groups (referred to as variant 1 (v.l), variant 2 (v.2), and variant 3 (v.3) in some reports (Masignani et al. 2003, supra) and Family A and B in other reports (see, e.g., Fletcher et al. 2004 Infect Immun 2088-2100)) based on amino acid sequence variability and immunologic cross-reactivity (Masignani et al.
  • leucine residue (L) at position 166 of the v.2 or v.3 fHBP sequence in Figure 3 refers to the residue at position 165 of the v.2 protein and at position 173 in the v.3 protein.
  • Figure 4 for alignment.
  • heterologous refers to two components that are defined by structures derived from different sources.
  • the chimeric polypeptide includes operably linked amino acid sequences that can be derived from different polypeptides (e.g., a first component from a fHBP v.l polypeptide and a second component from a fHBP v.2 polypeptide).
  • heterologous in the context of a polynucleotide encoding a chimeric polypeptide includes operably linked nucleic acid sequence that can be derived from different genes (e.g., a first component from a nucleic acid encoding a fHBP v.l polypeptide and a second component from a nucleic acid encoding a fHBP v.2 polypeptide).
  • Such chimeric polypeptides as described herein provide for presentation of epitopes in a single polypeptide that are normally found in different polypeptides.
  • heterologous nucleic acids include expression constructs in which a nucleic acid comprising a coding sequence is operably linked to a regulatory element (e.g., a promoter) that is from a genetic origin different from that of the coding sequence (e.g., to provide for expression in a host cell of interest, which may be of different genetic origin relative to the promoter, the coding sequence or both).
  • a regulatory element e.g., a promoter
  • a T7 promoter operably linked to a polynucleotide encoding a fHBP polypeptide or domain thereof is said to be a heterologous nucleic acid.
  • Heterologous in the context of recombinant cells can refer to the presence of a nucleic acid (or gene product, such as a polypeptide) that is of a different genetic origin than the host cell in which it is present.
  • a ⁇ eisserial amino acid or nucleic acid sequence of one strain is heterologous to a
  • Heterologous as used herein in the context of a chimeric fHBP (e.g.,
  • heterologous fHBP domain e.g., a “heterologous B domain”, “heterologous C domain” indicates that the chimeric fHBP protein contains operably linked and contiguous amino acid sequences of structural elements of at least two different fHBP variants (e.g., so as to provide for presentation of epitopes of a v.1 fHBP, and presentation of a v.2 fHBP and/or a v.3 fHBP in a single fHBP polypeptide).
  • a heterologous B domain refers to a polypeptide which comprises a B domain that contains a first portion having a contiguous amino acid sequence of a B domain of a first fHBP variant operably linked to a second portion having a contiguous amino acid sequence of a B domain of a second fHBP variant.
  • "Derived from” in the context of an amino acid sequence or polynucleotide sequence is meant to indicate that the polypeptide or nucleic acid has a sequence that is based on that of a reference polypeptide or nucleic acid (e.g., a naturally occurring fHBP protein or encoding nucleic acid), and is not meant to be limiting as to the source or method in which the protein or nucleic acid is made.
  • a reference polypeptide or nucleic acid e.g., a naturally occurring fHBP protein or encoding nucleic acid
  • “Derived from” in the context of bacterial strains is meant to indicate that a strain was obtained through passage in vivo, or in in vitro culture, of a parental strain and/or is a recombinant cell obtained by modification of a parental strain.
  • Constant amino acid substitution refers to a substitution of one amino acid residue for another sharing chemical and physical properties of the amino acid side chain (e.g., charge, size, hydrophobicity/hydrophilicity).
  • Consservative substitutions are intended to include substitution within the following groups of amino acid residues: gly, ala; val, ile, leu; asp, glu; asn, gin; ser, thr; lys, arg; and phe, tyr.
  • Conservative amino acid substitutions in the context of a chimeric fHBP disclosed herein are selected so as to preserve presentation of an epitope of interest.
  • the term "protective immunity” means that a vaccine or immunization schedule that is administered to a mammal induces an immune response that prevents, retards the development of, or reduces the severity of a disease that is caused by Neisseria meningitidis, or diminishes or altogether eliminates the symptoms of the disease.
  • Protective immunity can be accompanied by production of bactericidal antibodies. It should be noted that production of bactericidal antibodies against Neisseria meningitidis is accepted in the field as predictive of a vaccine's protective effect in humans. (Goldschneider et al., 1969, J. Exp.
  • a disease caused by a strain of capsular group B of Neisseria meningitidis encompasses any clinical symptom or combination of clinical symptoms that are present in an infection of a human with a member of capsular group B of Neisseria meningitidis. These symptoms include but are not limited to: colonization of the upper respiratory tract ⁇ e.g.
  • mucosa of the nasopharynx and tonsils by a pathogenic strain of capsular group B of Neisseria meningitidis, penetration of the bacteria into the mucosa and the submucosal vascular bed, septicemia, septic shock, inflammation, haemmorrhagic skin lesions, activation of fibrinolysis and of blood coagulation, organ dysfunction such as kidney, lung, and cardiac failure, adrenal hemorrhaging and muscular infarction, capillary leakage, edema, peripheral limb ischaemia, respiratory distress syndrome, pericarditis and meningitis.
  • the phrase "broad spectrum protective immunity” means that a vaccine or immunization schedule elicits "protective immunity” against at least more than one strain (and can be against at least two, at least three, at least four, at least five, against at least eight, or more strains) of Neisseria meningitidis, wherein each of the strains expresses a different fHBP subvariant or fHBP variant.
  • the present disclosure specifically contemplates and encompasses a vaccine or vaccination regimen that confers protection against a disease caused by a member of any capsular group (e.g., A, B, or C), with protection against disease caused by a capsular group B strain of Neisseria meningitidis being of interest due to the epidemiological prevalence of strains causing disease with this capsular group and lack of broadly effective group B vaccines.
  • a member of any capsular group e.g., A, B, or C
  • an antibody or “specifically immunoreactive with”, in the context of an antigen (e.g., a polypeptide antigen) refers to a binding reaction which is based on and/or is probative of the presence of the antigen in a sample which may also include a heterogeneous population of other molecules.
  • an antigen e.g., a polypeptide antigen
  • the specified antibody or antibodies bind(s) to a particular antigen or antigens in a sample and do not bind in a significant amount to other molecules present in the sample.
  • an antibody or “specifically immunoreactive with” in the context of an epitope of an antigen (e.g., an epitope of a polypeptide) refers to a binding reaction which is based on and/or is probative of the presence of the epitope in an antigen (e.g., polypeptide) which may also include a heterogeneous population of other epitopes, as well as a heterogeneous population of antigens.
  • the specified antibody or antibodies bind(s) to a particular epitope of an antigen and do not bind in a significant amount to other epitopes present in the antigen and/or in the sample.
  • Immune response indicators include but are not limited to: antibody titer or specificity, as detected by an assay such as enzyme- linked immunoassay (ELISA), bactericidal assay, flow cytometry, immunoprecipitation, Ouchter-Lowny immunodiffusion; binding detection assays of, for example, spot, Western blot or antigen arrays; cytotoxicity assays, etc.
  • ELISA enzyme- linked immunoassay
  • a "surface antigen” is an antigen that is present in a surface structure of
  • Neisseria meningitidis ⁇ e.g. the outer membrane, inner membrane, periplasmic space, capsule, pili, etc.).
  • Isolated refers to an entity of interest that is in an environment different from that in which the compound may naturally occur. "Isolated” is meant to include compounds that are within samples that are substantially enriched for the compound of interest and/or in which the compound of interest is partially or substantially purified.”
  • Enriched means that a sample is non-naturally manipulated (e.g., by an experimentalist or a clinician) so that a compound of interest is present in a greater concentration (e.g., at least a three-fold greater, at least 4-fold greater, at least 8-fold greater, at least 64-fold greater, or more) than the concentration of the compound in the starting sample, such as a biological sample (e.g., a sample in which the compound naturally occurs or in which it is present after administration), or in which the compound was made (e.g., as in a bacterial polypeptide, antibody, chimeric polypeptide, and the like)
  • a "knockout" of a gene involved in LPS synthesis indicates means that function of the gene has been substantially decreased so that the expression of the gene is not detectable or only present at insignificant levels and/or a biological activity of the gene product (e.g., an enzymatic activity) is significantly reduced relative to prior to the modification or is not detectable.
  • "Knock-outs” encompass conditional knock-outs, where alteration of the target gene can occur upon, for example, exposure to a predefined set of conditions (e.g., temperature, osmolarity, exposure to substance that promotes target gene alteration, and the like.
  • a “knock-in” or “knockin” of a target gene refers to a genetic alteration in a host cell genome that that results in an increase in a function provided by the target gene, FHBP AND FHBP-ENCODING NUCLEIC ACIDS
  • v.l fHBP of the N. meningitidis strain MC58 was arbitrarily selected as a reference sequence for all native v.l, v.2, and v.3 fHBP amino acid sequences, as well as for the chimeric fHBPs described herein.
  • Two nomenclature systems have been adopted to describe fHBP: one, which for convenience divided the protein into three domains, designated A, B and C (Giuliani et al., Infect Immun 2005; 73:1151-60), and the other based on three-dimensional structural data.
  • fHBP is divided into two domains: the fHbp ⁇ and the fHbpC. Details of each of these domains with reference to the amino acid sequence of v.l fHBP of MC58 strain is described below.
  • fHBP as having an "A domain", a "B domain", and a "C domain”.
  • the amino acid sequence of the v.l fHBP of the MC58 strain along with the boundaries of the A, B and C domains is shown in Fig. 21.
  • the QlOl and G164 residues indicated by the upward arrows denote the A/B and B/C domain boundaries, respectively.
  • the " ⁇ " symbols indicate the position of the first and second ⁇ helices of the fHBP (referred to as AHl and AH2).
  • Residues GEHT are underlined followed by the second ⁇ helix (AH2) of fHBP.
  • Panel A in Fig. 34 also shows a ribbon diagram of the full length fHBP with the A, B, and C domains indicated as various shades of gray.
  • FIG. 3 provides a schematic of a truncated structural model of fHBP having operably linked B and C domains (the A domain and a portion of the ⁇ -terminal portion of the B domain are not shown).
  • the native v.l fHBP of MC58 was again used as a reference sequence for purposes of residue numbering. Amino acid residues identified by site-directed mutagenesis of fHBP that contribute to binding of nine anti-fHBP mAbs (referred to as "JAR" mAbs) are noted. Coordinates from the solution structure of the B and C domains of fHBP from strain MC58 were used to construct the model.
  • the ⁇ helix of the B domain is illustrated, as are the loops and ⁇ strands of the C domain.
  • fHBP is described as having two structural domains as opposed to the three domains described above.
  • the two-domain nomenclature system is based on structural information of a full-length fHBP from which three-dimensional models may be constructed, such as the ones shown in Fig. 34. Structural modeling reveals that full-length fHBP is found to exist in solution as two separate domains connected by a linker.
  • the amino acid sequence of the v.l fHBP of the MC58 strain is shown in Fig. 21 with end of the fHbpN domain indicated with an arrow at glycine 136.
  • the N- terminal domain is named fHbpN (residues 8-136) and the C-terminal domain fHbpC (residues 141-255), each comprising at least 8 antiparallel ⁇ strands and joined by a native linker (residues 137-140).
  • the linker also comprises ⁇ -helix AH2 as " ⁇ " below the sequence in Fig. 21 marks the positions of ⁇ helices that reside in fHBP.
  • the fHbpC domain is considered to include the linker that connects the N-terminal and C-terminal domains based on the convention of this nomenclature.
  • fHBP has been divided into three variant groups (referred to as variant 1 (v.l), variant 2 (v.2), and variant 3 (v.3)) based on amino acid sequence variability and immunologic cross-reactivity (Masignani et al. 2003 J Exp Med 197:789-99).
  • fHBP has also been subdivided into two sub-families designated sub-family A (which includes v.2 and v.3 of Masignani et al., 2003 J Exp Med 197:789-99) and sub-family B (v.l) (Fletcher et al., 2004, Infect Immun. 72: 2088-100).
  • Variant as used in the context of an "fHBP variant” refers to an fHBP that share at least 89% amino acid sequence identity with the prototype strain of that variant group (strain MC58 for v.l; strain 2996 for v.2; and strain M1239 for v.3). These were the original prototype sequences described by Masignani et al., J. Exp. Med., 2003. Strains within a variant group encode fHBPs with greater than 88% amino acid identity, whereas strains of different fHBP variant groups range from approximately 60-88% identical.
  • fHBPs in the same "variant" group possess greater than 88% identity to the respective prototype sequence (v.l, strain MC58; v.2, strain 2996; v.3, strain M1239).
  • a "subvariant" as used in the context of an "fHBP subvariant” refers to fHBP polypeptides that differ from the prototype sequence.
  • strain NZ98/254 is referred to as an fHBP v.1 subvariant, with 91% identity to the prototype sequence from strain MC58; strain RM1090 is referred to as an fHBP v.2 subvariant, with a sequence that is 94% identical to the v.2 prototype strain 2996.
  • fHBP polypeptides, and encoding nucleic acids, from which portions of the chimeric fHBPs of the present disclosure can be derived may be from any suitable N. meningitidis strain.
  • N. meningitidis strains are divided into serologic groups (capsular groups), serotypes (PorB phenotypes) and subtypes (PorA phenotypes) on the basis of reactions with polyclonal (Frasch, C. E. and Chapman, 1973, /. Infect. Dis.
  • Capsular grouping traditionally has been based on immunologically detectable variations in the capsular polysaccharide but is being replaced by PCR of genes encoding specific enzymes responsible for the biosynthesis of the structurally different capsular polysaccharides. About 12 capsular groups (including A, B, C, X, Y, Z, 29-E, and W- 135) are known. Strains of the capsular groups A, B, C, Y and W- 135 account for nearly all meningococcal disease. Serotyping traditionally has been based on monoclonal antibody defined antigenic differences in an outer membrane protein called Porin B (PorB).
  • Porin B Porin B
  • Serosubtyping has been based on antibody defined antigenic variations on an outer membrane protein called Porin A (PorA). Both serotyping and serosubtyping are being replaced by PCR and/or D ⁇ A sequencing for identification of genes encoding the variable regions of PorB and PorA, respectively that are associated with mAb reactivity (e.g. Sacchi, Lemos et al., supra; Urwin et al., 1998, Epidem. and Infect. 120:257).
  • N. meningitidis also may be divided into clonal groups or subgroups, using various techniques that directly or indirectly characterize the bacterial genome. These techniques include multilocus enzyme electrophoresis (MLEE), based on electrophoretic mobility variation of an enzyme, which reflects the underlying polymorphisms at a particular genetic locus. By characterizing the variants of a number of such proteins, genetic "distance" between two strains can be inferred from the proportion of mismatches. Similarly, clonality between two isolates can be inferred if the two have identical patterns of electrophoretic variants at number of loci.
  • MLEE multilocus enzyme electrophoresis
  • MLST multilocus sequence typing
  • the specification provides the amino acid sequence of exemplary fHBPs from which the chimeric fHBP can be derived, this is not intended to be limiting.
  • the chimeric fHBP can contain amino acid sequences that are at least 80%, at least 85%, at least 90%, or at least 95% identical to an amino acid sequence of a naturally- occurring fHBP.
  • Nucleic acids encoding fHBP polypeptides for use in construction of chimeric fHBPs contemplated herein are known in the art. Exemplary fHBP polypeptides are described in, for example, WO 2004/048404; Masignani et al. 2003 J Exp Med 197:789-799; Fletcher et al. Infect Immun 2004 2088-2100; Welsch et al. J Immunol 2004 172:5606-5615; and WO 99/57280. Nucleic acid (and amino acid sequences) for fHBP variants and subvariants are also provided in GenBank as accession nos.: NC_003112, GenelD: 904318 (NCBI Ref.
  • NP_274866 (from N. meningitidis strain MC58); AY548371 (AAT01290.1) (from N. meningitidis strain CU385); AY548370 (AAT01289.1) (from N. meningitidis strain H44/76); AY548377 (AAS56920.1) (from N. meningitidis strain M4105); AY548376 (AAS56919.1) (from N. meningitidis strain M1390); AY548375 (AAS56918.1) (from N. meningitidis strain ⁇ 98/254); AY548374 (AAS56917.1) (from N.
  • the immature fHBP protein includes a leader sequence of about 19 residues.
  • the ordinarily skilled person can readily envision the sequences of nucleic that can encode for, and provide for expression of, a polypeptide having such an amino acid sequence.
  • polypeptides and nucleic acids having sequences that are at least 80%, at least 85%, at least 90%, or at least 95% identical in sequence to such exemplary amino acid and nucleic acids.
  • nucleic acid sequences refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (e.g., at least 80%, at least 85%, at least 90%, or at least 95% identical over a specified region), when compared and aligned for maximum correspondence over a designated region, e.g., a B domain or portion thereof, e.g., a region at least about 30, 35, 40, 45, 50, 55, 60, 65 or more amino acids or nucleotides in length, and can be up to the full-length of the reference amino acid or nucleotide sequence (e.g., a full-length fHBP).
  • the disclosure specifically contemplates both naturally-occurring polymorphisms and synthetically produced amino acid sequences and their encoding nucleic acids.
  • test and reference sequences are input into a computer program, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • Examples of algorithms that are suitable for determining percent sequence identity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. MoI. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389- 3402, respectively.
  • Software for performing BLAST analyses is publicly available through 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.
  • residue positions which are not identical differ by conservative amino acid substitutions.
  • Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine
  • a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine
  • a group of amino acids having amide-containing side chains is asparagine and glutamine
  • a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan
  • a group of amino acids having basic side chains is lysine, arginine, and histidine
  • a group of amino acids having sulfur-containing side chains is cysteine and methionine.
  • Sequence identity between two nucleic acids can also be described in terms of hybridization of two molecules to each other under stringent conditions.
  • the hybridization conditions are selected following standard methods in the art (see, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N. Y.).
  • An example of stringent hybridization conditions is hybridization at 5O 0 C or higher and 0.1 x SSC (15 mM sodium chloride/1.5 mM sodium citrate).
  • Stringent hybridization conditions are hybridization conditions that are at least as stringent as the above representative conditions, where conditions are considered to be at least as stringent if they are at least about 80% as stringent, typically at least 90% as stringent as the above specific stringent conditions.
  • fHBP may be described as having the following three domains to facilitate analysis: A domain, B domain, and C domain.
  • a domain As shown in Fig. 21, the upward arrows at QlOl and G164 demarcate the boundaries between A/B domains and B/C domains, respectively.
  • the chimeric fHBPs of the present disclosure optionally include an A domain.
  • the A domain can be structurally defined as those residues corresponding to residues 1-100 of v.l fHBP of MC58, where the numbering is based on amino acid sequence of MC58 v.l fHBP lacking the signal sequence (Masignani et al., 2003 J Exp Med 197:789-99) (see Figure 21).
  • Chimeric fHBPs which contain an A domain can contain a contiguous A domain amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to an amino acid sequence of an A domain of a naturally occurring fHBP.
  • the A domain may be derived from the same variant group (and may be derived from the same fHBP) as the N-terminal portion of the B domain, such that the amino acid sequence at the A/B junction is one that may be found in nature.
  • the A domain amino acid sequence may be derived from a fHBP variant group different from the fHBP variant group from which the N-terminal amino acid sequence of the B domain is derived (e.g., the A domain may be derived from a v.2 fHBP and the N-terminal amino acid sequence of the B domain derived from a v.l fHBP).
  • the chimeric fHBPs of the present disclosure contain amino acid sequence of a v.l fHBP B domain.
  • Amino acid sequences of v.l fHBP, including v.l fHBP B domains, are well known in the art and can be used to derive the desired amino acid sequence of a chimeric fHBP disclosed herein.
  • Fig. 13 provides the amino acid sequences of an N-terminal portion of the B domain of selected v.1 fHBPs. The alignment illustrates the position and identity of naturally occurring polymorphisms among v.l fHBPs.
  • Figure 8 A illustrates the amino acid sequence identity between full length fHBPs of exemplary v.l, v.2 and v.3 strains, and further illustrates the presence or absence of epitopes defined by the indicated JAR mAbs.
  • Figure 8B illustrates the amino acid sequence identity between the A, B, and C domains of exemplary v.l, v.2 and v.3 fHBPs, as well as amino acid sequence identity within the N-terminal (101-135) and C-terminal (136-164) portions of B domains. [0087] Fig.
  • FIG. 21 shows the amino acid sequence of the fHBP of the v.l strain MC58, and illustrates the position and of a full-length B domain (defined by residues 101-164 and encompassing the amino acid sequence of GEHT followed by ⁇ -helix AH2).
  • Panel A illustrates that an N-terminal portion of the B domain of the v.1 fHBP of the MC58 strain, can encompass an amino acid sequence defined by residues corresponding to residues N- terminal of the GEHT residues and extending to the N-terminus of the B domain at a residue corresponding to residue 101.
  • the C-terminal portion of the B domain of the v.1 fHBP of the MC58 strain can encompass an amino acid sequence defined by those residues corresponding to an amino acid sequence extending N-terminally from residue 164 of the B domain, and encompassing up to and including the amino acid sequence of GEHT.
  • a full-length B domain is structurally defined by the residues corresponding to residues 101-164 of the v.l of fHBP of MC58, where residues 101-135 can define an exemplary N-terminal portion of the B domain and residues 136-164 can define an exemplary C-terminal portion of the B domain, where the numbering is based on amino acid sequence of MC58 v.l fHBP lacking the signal sequence (Masignani et al., 2003 J Exp Med 197:789-99).
  • the C- terminus of the N-terminal portion of the B domain (and thus the N-terminus of the C- terminal portion of the heterologous B domain) is defined by the position of the junction point, which can be present N-terminal or C-terminal to the amino acid sequence of GEHT, as discussed below in more detail.
  • the junction point of a heterologous B domain of a chimeric fHBP can be positioned C-terminal to a sequence corresponding to the GEHT, and thus can extend beyond a residue corresponding to residue 135.
  • Exemplary chimeric fHBP include those comprising a B domain having a contiguous amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to an N-terminal B domain amino acid sequence of a v.l fHBP, e.g., at least 80%, at least 85%, at least 90%, or at least 95% identical to a contiguous amino acid sequence of the N-terminal B domain amino acid sequence exemplified in Fig. 13.
  • Exemplary chimeric fHBP having a heterologous B domain contain at least 35, at least 40, at least 45, at least 50 residues (and in some embodiments no more than 50 residues) of a contiguous N-terminal amino acid sequence of a B domain of a v.l fHBP.
  • Exemplary chimeric fHBPs of the present disclosure contain a heterologous B domain containing an N-terminal amino acid sequence derived from an N-terminal portion of a v.1 fHBP B domain and the remaining C-terminal portion derived from the corresponding C-terminal portion of a v.2 (or v.3) fHBP B domain, followed by the contiguous amino acid sequence of a v.2 (or v.3) C domain.
  • the C domain can be structurally defined as those residues corresponding to residues 165-255 of v.l fHBP of MC58, where the numbering is based on amino acid sequence of MC58 v.l fHBP lacking the signal sequence (Masignani et al., 2003 J Exp Med 197:789-99) (see Figure 21).
  • chimeric fHBP are well known in the art and can be used to derive the desired amino acid sequence of a chimeric fHBP disclosed herein.
  • Fig. 14 provides the amino acid sequences of a C-terminal portion of the B domain (as exemplified by the C-terminal 25 amino acids of v.2 fHBP B domain) and the full-length C domains of selected v.2 fHBPs.
  • the alignment illustrates the position and identity of naturally occurring polymorphisms among v.2 fHBPs.
  • Exemplary chimeric fHBP include those comprising a B domain containing a
  • C-terminal amino acid sequence derived from a v.2 or v.3 B domain usually having a contiguous amino acid sequence that is greater than or at least 85%, at least 90%, or at least 95% identical to an C-terminal B domain amino acid sequence of a v.2 or v.3 fHBP, e.g., at least 80%, at least 85%, at least 90%, or at least 95% identical to a contiguous amino acid sequence of the N-terminal B domain amino acid sequence, such as those v.2 sequences exemplified in Fig. 14.
  • the B domain can be at least 80%, at least 85%, at least 90%, or at least 95% identical to a contiguous amino acid sequence of a full-length B domain amino acid sequence of a v.1 fHBP.
  • a full-length B domain of a v.l fHBP generally is about 64 residues in length.
  • fHBP may also be described has having two structural domains: fHbpN and fHbpC, based on an alternative nomenclature that is derived from the structure of the full-length fHBP.
  • glycine 136 marks approximately the beginning of a linker between the N-terminal and the C- terminal domains, named the fHbpN and fHbpC domains, respectively.
  • the chimeric fHBP of the present disclosure may include a full-length fHbpN domain or a partial fHbpN domain.
  • the fHbpN domain can be structurally defined as those residues corresponding to residues 1-136 of v.l fHBP of MC58, where the numbering is based on amino acid sequence of MC58 v.l fHBP lacking the signal sequence (Masignani et al., 2003 J Exp Med 197:789-99) (see Fig. 21).
  • the respective amino acid sequences of the first 100 residues of the fHbpN domain normally share significant amino acid sequence identity (Fig. 8B).
  • Chimeric fHBP which contains an fHbpN domain can contain a contiguous fHbpN domain amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to an amino acid sequence of an fHbpN domain of a naturally occurring fHBP.
  • the chimeric fHBP may include a partial fHbpN domain, such that an N-terminal portion of the fHbpN is truncated.
  • the partial fHbpN domain may comprise at least 30, 40, or 50 of a contiguous C-terminal amino acid sequence of the full length fHbpN domain.
  • Exemplary chimeric fHBPs include those comprising a full or partial fHbpN domain having a contiguous amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to at least a C-terminal portion of the fHbpN amino acid sequence of a v.l fHBP, e.g., at least 80%, at least 85%, at least 90%, or at least 95% identical to a contiguous amino acid sequence of the C-terminal fHbpN amino acid sequence exemplified in Fig. 13.
  • An exemplary chimeric fHBP having heterologous domains contains at least 35, at least 40, at least 45, at least 50 residues (and in some embodiments no more than 50 residues) of a contiguous C-terminal amino acid sequence of an fHbpN domain of a v.l fHBP.
  • the full or partial fHbpN domain may be derived from the same variant group
  • the fHbpN amino acid sequence may be derived from a fHBP variant group different from the fHBP variant group from which the N-terminal amino acid sequence of the fHbpC domain is derived (e.g., the fHbpC domain may be derived from a v.2 fHBP and the C-terminal amino acid sequence of the fHbpN domain derived from a v.l fHBP).
  • the chimeric fHBPs of the present disclosure contain amino acid sequence of a v.l fHbpN domain.
  • Amino acid sequences of v.l fHBP are well known in the art and can be used to derive the desired amino acid sequence of a chimeric fHBP disclosed herein.
  • Fig. 13 provides the C-terminal amino acid sequences of the fHbpN domain of selected v.l fHBPs. The alignment illustrates the position and identity of naturally occurring polymorphisms among v.l fHBPs.
  • Figure 8A illustrates the amino acid sequence identity between full length fHBPs of exemplary v.l, v.2 and v.3 strains, and further illustrates the presence or absence of epitopes defined by the indicated JAR mAbs.
  • Figure 8B illustrates the amino acid sequence identity between exemplary v.l, v.2 and v.3 fHBPs, as well as amino acid sequence identity within the C-terminal portion of the fHbpN (101-135) and N-terminal (136-164) portion of the fHbpC domains.
  • the position of the junction point between heterologous domains can be present within or proximal to the linker that connects the fHbpN and the fHbpC domains.
  • Glycine 136 defines the boundary between fHbpN and fHbpC and also marks the beginning of the linker sequence.
  • the linker sequence corresponds approximately to residues 136 to 149 and includes ⁇ -helix AH2.
  • the junction point between heterologous domains of a chimeric fHBP can be positioned C-terminal to a sequence corresponding to the GEHT sequence underlined in Fig. 21.
  • the junction may be no more than 20, no more than 15, no more than 5 or less amino acid residues away from the amino acid sequence of GEHT or the linker sequence.
  • the junction between the heterologous domains may be positioned C-terminal to glycine 164.
  • Glycine 164 is also indicated by an arrow in Fig. 21.
  • Exemplary chimeric fHBPs of the present disclosure contain heterologous domains comprising a full or partial fHbpN domain of a v.l fHBP fHbpN domain and a fHbpC domain derived from the fHbpC of a v.2 (or v.3) fHBP.
  • the fHbpC domain can be structurally defined as those residues corresponding to residues 141-255 of v.l fHBP of MC58, where the numbering is based on amino acid sequence of MC58 v.l fHBP lacking the signal sequence (Masignani et al., 2003 J Exp Med 197:789-99) (Fig. 21).
  • Amino acid sequences of v.2 and v.3 fHBP including v.2 and v.3 fHBP fHbpN and fHbpC domains, are well known in the art and can be used to derive the desired amino acid sequence of a chimeric fHBP disclosed herein.
  • Fig. 14 provides the amino acid sequences of the full-length fHbpC domains of selected v.2 fHBPs. The alignment illustrates the position and identity of naturally occurring polymorphisms among v.2 fHBPs.
  • Exemplary chimeric fHBPs include those comprising an fHbpC amino acid sequence derived from a v.2 or v.3 fHbpC domain, usually having a contiguous amino acid sequence that is greater than or at least 85%, at least 90%, or at least 95% identical to the amino acid sequence of the fHbpC domain of a v.2 or v.3 fHBP, e.g., at least 80%, at least 85%, at least 90%, or at least 95% identical to a contiguous amino acid sequence of the fHbpC domain amino acid sequence, such as those v.2 sequences exemplified in Fig. 14.
  • fHbpC domain may contain two contiguous amino acid sequences derived from different variants.
  • a contiguous N-terminal amino acid sequence of fHbpC can be at least 80%, at least 85%, at least 90%, or at least 95% identical to a contiguous amino acid sequence of the corresponding amino acid sequence of a v.l fHBP.
  • fHBP may be described in the context of the three domains assigned by Giuliani et al (Infect Immun 2005 ; 73 : 1151 -60) or in the context of two three-dimensional structural domains.
  • the disclosure will adopt the nomenclature of the three domains, designated A, B, and C domains.
  • A, B, and C domains the chimeric fHBPs of the present disclosure.
  • the chimeric fHBPs of the present disclosure generally include either a heterologous B domain and a C domain; or a B domain and a heterologous C domain.
  • chimeric fHBPs are constructed so as to contain epitopes that elicit bactericidal antibodies effective against N. meningitidis strains producing more than one fHBP variant.
  • the term "chimeric factor H binding protein" or "chimeric fHBP” refers to a polypeptide comprising, from ⁇ -terminus to C-terminus, an amino acid sequence of a B domain and of a C domain, wherein at least one of the B domain and the C domain contains a heterologous amino acid sequence characterized as having an ⁇ -terminal portion derived from a contiguous amino acid sequence of a v.l fHBP with the remaining B and C-terminal portion (or C terminal portion) being derived from a contiguous amino acid sequence of a v.2 or v.3 fHBP.
  • the B domain and/or C domain amino acid sequences are generally derived from a contiguous amino acid sequence of a naturally-occurring fHBP and mutants thereof that maintain or introduce desired epitopes.
  • Chimeric fHBP can optionally include an amino acid sequence of an fHBP A domain operably linked and ⁇ -terminal to the B domain.
  • Chimeric fHBP can further optionally include a leader sequence, e.g., to provide for expression of the chimeric fHBP on a cell surface of a bacterial host cell.
  • B domain generally comprises at least an ⁇ -terminal portion derived from a contiguous amino acid sequence of a v.l fHBP B domain and a C-terminal portion derived from a contiguous amino acid sequence of a v.2 or v.3 B domain, with the heterologous B domain being operably linked to a C domain derived from a contiguous amino acid sequence of a v.2 or v.3 fHBP C domain.
  • such chimeric fHBP can be described as having a heterologous B domain composed of an ⁇ -terminal portion for which a corresponding contiguous amino acid sequence of a v.l fHBP B domain sequence serves as a scaffold, and a C-terminal portion for which a corresponding contiguous amino acid sequence of a v.2 or v.3 fHBP B domain sequence serves as a scaffold.
  • exemplary chimeric fHBP having a heterologous B domain contain at least 35, at least 40, at least 45, at least 50 residues (and in some embodiments no more than 50 residues) of a contiguous ⁇ -terminal amino acid sequence of a B domain of a v.l fHBP.
  • the B domain of the chimeric fHBP comprises a contiguous amino acid sequence of a v.l fHBP B domain operably linked to heterologous C domain comprising at least an ⁇ -terminal portion of a v.1 fHBP C domain and a C-terminal portion of a v.2 or v.3 C domain.
  • Exemplary chimeric fHBP of this embodiment contain 2, 4, 6, 8 residues of an N-terminal sequence of a v.l C domain, with the remainder of the C domain being derived from a v.2 or v.3 C domain amino acid sequence.
  • Chimeric fHBP contemplated by the present disclosure include those having an amino acid sequence corresponding to a full-length B domain and a full-length C domain, and, optionally, a full-length A domain wherein the chimeric fHBP includes at least a heterologous B domain or a heterologous C domain.
  • Other embodiments include chimeric fHBP having an amino acid sequence corresponding to a fragment of an A domain composed of a contiguous amino acid sequence encompassing amino acid defining an epitope bound by the JAR 4 mAb.
  • chimeric fHBP in which the C domain is truncated at the C-terminus, with the proviso that epitopes of interest (e.g., one or more of the epitopes bound by mAbs JAR 10, JAR 11, JAR 33, JAR 32/35, and JAR 13) are preserved so as to retain the ability to elicit antibodies that bind these epitopes.
  • Chimeric fHBP also include those that lack an A domain, and have an N-terminally truncated B domain, with the proviso that the truncated B domain maintains expression of an epitope(s) of interest.
  • Chimeric fHBP include those having a B domain that expresses an epitope bound by the JAR 5 mAb.
  • Chimeric polypeptides described herein can include additional heterologous amino acid sequences, e.g., to provide an N-terminal methionine or derivative thereof (e.g., pyroglutamate) as a result of expression in a bacterial host cell (e.g., E. coli) and/or to provide a chimeric polypeptide having a fusion partner at its N-terminus or C-terminus.
  • Fusion partners of interest include, for example, glutathione-S-transferase (GST), maltose binding protein (MBP), His 6 -tag, and the like, as well as leader peptides from other proteins, particularly lipoproteins. Fusion partners can provide for additional features, such as in facilitating isolation and purification of the chimeric polypeptide.
  • Native fHBP usually contains an N-terminal cysteine to which a lipid moiety can be covalently attached.
  • This cysteine residue is usually lipidated in the naturally- occurring protein, and can be lipidated in the chimeric fHBPs disclosed herein.
  • reference to "cysteine" or "C” at this position specifically includes reference to both an unmodified cysteine as well as to a cysteine that is lipidated (e.g., due to post-translational modification).
  • the chimeric fHBP can be lipidated or non-lipidated.
  • lipidated fHBP previously has been purified from the membrane fraction of E. coli protein by detergent extraction (Fletcher et al., 2004 Infection and Immunity 72(4):2088-100), which method may be adapted for the production of lipidated chimeric fHBP.
  • Lipidated proteins may be of interest as such can be more immunogenic than soluble protein (see, e.g., Fletcher et al., 2004 Infection and Immunity 72(4):2088-100).
  • Exemplary chimeric fHBPs are described in detail below.
  • the chimeric fHBPs of the present disclosure encompass those that can be described in terms of one or more of, for example, the site at which heterologous sequences are joined within the chimeric fHBP (i.e., the "junction point"), the presence of epitopes specifically bound by a mAb, amino acid sequence, or any combination of such features that may be present in exemplary fHBPs.
  • the junction point of the chimeric fHBP is the point at which amino acid sequence of the chimeric fHBP shifts from being derived from a contiguous amino acid sequence of a v.l fHBP to being derived from contiguous amino acid sequence of a v.2 or v.3 fHBP.
  • the junction point thus provides for an amino acid sequence that is heterologous, i.e., derived from different fHBPs.
  • heterologous domain i.e., heterologous B domain or heterologous C domain
  • junction point thus defining the length of the N- terminal and C-terminal portions of the chimeric domain that are derived from a v.1 or v.2/v.3 amino acid sequence.
  • a B domain amino acid sequence comprising an amino acid sequence N-terminal to the second ⁇ helix of fHBP, which includes residues corresponding to those implicated in defining the JAR 5 mAb epitope (i.e., residues at positions 121 and 122 of a B domain v.l fHBP MC58, which are glycine and lysine, respectively) is denoted as the "N-terminal portion of the B domain" (see, e.g., Fig. 13, Fig. 21 and Fig. 22, Panel A).
  • the amino acid sequence flanking and C-terminal to the N-terminal portion of the B domain is the "C-terminal (or distal) portion of the B domain" and is derived from a contiguous amino acid sequence of a v.2 or v.3 fHBP (Fig. 14 and Fig. 22). Together, the N-terminal and C-terminal portions of the B domain compose a heterologous B domain of a chimeric fHBP of the present disclosure.
  • the junction point may be positioned at a residue adjacent to the second ⁇ helix (AH2) (e.g., adjacent and C- terminal to a residue corresponding to residue 121 or 122 of Figure 21, e.g., adjacent and C- terminal to one of the residues of GEHTSFDK, e.g., adjacent and C-terminal to one of the residues of GEHT, N-terminal to AH2), or at a position C-terminal to AH2.
  • AH2 residue adjacent to the second ⁇ helix
  • the junction point of the heterologous B domain can be positioned at any site corresponding to a site after the glycine residue or after the lysine residue, that define a JAR 5 monoclonal antibody (mAb) epitope of a v.1 fHBP (which residue is positioned within the B domain, i.e., at G121 or K122 of v.l fHBP strain MC58 reference sequence) but before a residue corresponding to a residue defining a JAR 11 mAb epitope of a v.2 fHBP (which residue is positioned in the C domain, i.e., A174 of v.2 fHBP strain 8047 reference sequence).
  • mAb monoclonal antibody
  • the heterologous B domain is provided such that the JAR 5 mAb epitope, the JAR 11 epitope, or both the JAR 5 and JAR 11 epitopes are maintained such that the chimeric fHBP is specifically bound by the respective mAb.
  • the junction point is positioned so that the chimeric fHBP contains a heterologous B domain, which has an N-terminal portion composed of a contiguous amino acid sequence of an N-terminal portion of a B domain of a v.1 fHBP containing a JAR 5 epitope (defined in part by G121 of v.l fHBP strain MC58) with the remaining portion (i.e., the C-terminal portion) of the B domain derived from a contiguous amino acid sequence of the corresponding C-terminal portion of a v.2 or v.3 fHBP B domain.
  • a heterologous B domain which has an N-terminal portion composed of a contiguous amino acid sequence of an N-terminal portion of a B domain of a v.1 fHBP containing a JAR 5 epitope (defined in part by G121 of v.l fHBP strain MC58) with the remaining portion (i.e., the C-terminal portion
  • the heterologous B domain is operably linked to a C domain derived from a contiguous amino acid sequence of a v.2 or v.3 fHBP, which can be the same or different v.2 or v.3 fHBP as that from which the C-terminal portion of the B domain is derived.
  • Exemplary heterologous B domains include those at least 80% identical, at least 85% identical, at least 90% identical, at least 99% identical or more to a contiguous amino acid sequence of a v.l fHBP corresponding to residues 101-121, 101-122, 101-123, 101-124, 101-125, 101-126, 101-127, 101-128, 101-129, 101-130, 101-131, 101-132, 101- 133, 101-134, 101-134, 101-136, 101-137, 101-138, or 101-139 of a v.l fHBP amino acid sequence, where the numbering is based on MC58 fHBP as a reference.
  • Such heterologous B domains include those having an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 99% identical or more to a contiguous amino acid sequence of a v.2 or v.3 fHBP so as to provide the remainder of the heterologous B domain having a C-terminus corresponding to residue 164 (again, using MC58 fHBP as a reference sequence of purposes of numbering).
  • the C-terminal portion of the heterologous B domain includes residues 123-164 of a v.2 or v.3 fHBP.
  • the C-terminal portion of the heterologous B domain can include an amino acid sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 99% identical or more to a contiguous amino acid sequence of a v.2 or v.3 fHBP corresponding to residues 122-164, 123-164, 124-164, 125-164, 126-164,127-164, 128-164, 129-164, 130-164, 131—164, 132-164, 133-164, 134-164, 135-164, 136-164, 137- 164, 139-164, or 140-164, where the N-terminal portion of the heterologous B domain is provided by the v.l sequences exemplified above.
  • the junction point is positioned N-terminal to the second ⁇ helix (AH2), which are denoted in Figure 22 by " ⁇ ".
  • AH2 second ⁇ helix
  • the residues GEHT are highly conserved across v.l, v.2, and v.3 fHBP variants, and thus can serve as convenient junction point residues, as well as a convenient reference for the position of a junction point in a chimeric fHBP.
  • the junction point can be positioned within 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 residue(s) N-terminal to GEHT to provide a heterologous B domain (e.g., positioned at a site not more than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 residue N-terminal of GEHT); or is positioned within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 residues C-terminal to GEHT (e.g., positioned at a site not more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 residues C-terminal of GEHT), where a junction point at a site less than or equal to 26 residues C-terminal to GEHT provides a heterologous B domain and a junction point positioned at more than 26 residues C-terminal to GEHT produces
  • the junction point of the heterologous B domain can be selected such that the heterologous B domain amino acid sequence positioned N-terminal and flanking the amino acid sequence GEHT is derived from a v.l fHBP B domain amino acid sequence and the heterologous B domain amino acid sequence positioned C-terminal and flanking the GEHT is derived from a v.2 or v.3 fHBP B amino acid sequence.
  • the junction point is positioned so as to provide a heterologous B domain comprising an amino acid sequence that is greater than 80% (e.g., at least 81%), at least 85%, at least 90%, at least 95% or identical to an amino acid sequence of
  • QSHSALTAFQ TEQIQDSEHS GK (SEQ ID NO: 1) where the amino acid sequence optionally provides for an epitope that mediates specific binding of a JAR 5 mAb.
  • Exemplary amino acid substitutions of the above sequence are as follows: QSHSALTA(F/L)Q TEQ(I/V/E)QD(S/P)E(H/D)S (GZEZR)K.
  • amino acid sequences of the heterologous B domain as set out above include, for example, one or more of the following substitutions of
  • SEQ ID NO:1 as follows: leucine (L) for the phenylalanine (F) at a residue corresponding to position 9; valine (V) or glutamic acid (E) for isoleucine (I) at residue position 14; proline (P) for serine (S) at residue position 17; aspartic acid (D) for histidine (H) at residue position 19; arginine (R) for glutamine (Q) at residue position 28; valine (V) for alanine (A) at residue position 35; glycine (G) for aspartic acid (D) at residue position 42; or lysine (K) for glutamic acid (E) at residue position 46.
  • heterologous B domain comprises an amino acid sequence represented by the formula:
  • the junction point is positioned so as to provide a heterologous B domain comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% or identical to an amino acid sequence of
  • TEQIQDSEHS GKMVAKRQFR IGDIAGEHTA FNQLPD where the amino acid sequence optionally provides for an epitope that mediates specific binding of a JAR 5 mAb.
  • the heterologous B domain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% or identical to an amino acid sequence of
  • the heterologous B domain comprises a sequence at least 80%, at least 85%, at least 90%, at least 95% or identical to an amino acid sequence set out in Fig. 13, which provides an alignment of exemplary chimeric fHBPs III, IV and V sequences in the region of the junction point, which is indicated by the box.
  • the residue, G121, implicated in the JAR 5 epitope is shown in bold and underlined.
  • the junction point is positioned so as to provide a heterologous B domain comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% or identical to an amino acid sequence of:
  • LTAFQ TEQIQDSEHS GKMVAKRQFR IGDIA where the amino acid sequence optionally provides for an epitope that mediates specific binding of a JAR 5 mAb.
  • the junction point of the chimeric fHBP is positioned so that the chimeric fHBP contains a heterologous C domain composed of a contiguous amino acid sequence of an N-terminal portion of a C domain of a v.1 fHBP and a contiguous amino acid sequence of a C-terminal portion of a C domain of a v.2 or v.3 fHBP.
  • the junction point of a chimeric fHBP having a heterologous C domain can be in the loop regions of the ⁇ -barrel of the C domain, or in any highly conserved segment, for example at residues D160 or 1170.
  • the heterologous C domain includes an N-terminal sequence of KLTYTIDFA.
  • Exemplary chimeric fHBP are provided in the Examples below.
  • the present disclosure contemplates these exemplary chimeric fHBP, as well as chimeric fHBP having at least 85%, at least 90%, at least 95% or greater amino acid sequence identity to the amino acid sequences of these exemplary chimeric fHBP (e.g., Chimera I, Chimera II, Chimera lib, Chimera III, Chimera IV, and Chimera V).
  • the amino acid and nucleic acid sequences encoding Chimera I, Chimera II, Chimera lib, Chimera III, Chimera IV, and Chimera V are provided in Figures 23, 24, 25, 26, 27, and 28, respectively.
  • the asterisk denotes the C- terminus of the amino acid sequence (corresponding to the stop codon of the encoding nucleic acid).
  • Chimeric fHBP encompass chimeric fHBP that contain pairs of epitopes that elicit antibodies that, when both are bound to their respective epitopes, exhibit enhanced bactericidal activity against N. meningitidis than when either one is bound alone.
  • Chimeric fHBP can be designed so as to ensure that such epitopes are maintained or to introduce such epitopes (e.g., by modification of an fHBP amino acid sequence to include a pair of epitopes heterologous to that fHBP amino acid sequence).
  • the distance between epitopes of such epitope pairs is selected so as to be less than 27 A but more than 14 A, and are usually located within a distance of about 18 A - 20 A.
  • a greater bactericidal effect was observed when two antibodies bound epitopes located at distances within these parameters. Without being held to theory, when bound by their respective antibodies, the distance between the epitopes is sufficient to facilitate interaction of the antibodies with factors of the complement cascade, but not so close as to result in inhibition of binding due to steric hindrance.
  • Chimeric fHBP containing such epitopes for v.2 and v.3 strains can thus provide for production of antibodies having greater bactericidal activity against such strains.
  • epitope pairs are those epitopes bound by JAR 10 and JAR 11 (fHBP v.2); and by JAR 33 and JAR 32/ 35 (JAR 32 and JAR 35 bind to the same or overlapping epitopes) (fHBP v.3).
  • Chimeric fHBPs can also include epitope pairs where one epitope of the pair is defined by binding by the mAb JAR 4 and the second epitope is bound by an antibody that inhibits fH binding.
  • Such an epitope pair is not necessarily subject to the constraints on distance between the epitopes as discussed above, with the proviso that the epitopes are not so close as to inhibit binding of their respective antibodies.
  • binding of an antibody that inhibits fH binding can be bactericidal along with another mAb that does not inhibit fH binding.
  • a mAb pair such as JAR 4 along with any anti-v.l or v.2 mAb that blocks fH binding can provide for production of antibodies that have enhanced bactericidal activity.
  • Chimeric fHBPs can be designed so as to include an epitope(s) that elicits antibodies that, when bound to fHBP, inhibit fH binding.
  • an epitope(s) that elicits antibodies that, when bound to fHBP, inhibit fH binding.
  • JAR 13 v.2 epitope
  • JAR 11 v.2 epitope
  • JAR 32/35 v.3 epitope
  • the chimeric fHBP can be generated using recombinant techniques to manipulate nucleic acids of different fHBPs known in the art to provide constructs encoding a chimeric fHBP of interest.
  • nucleic acids encoding a variety of different v.l, v.2, and v.3 fHBPs of N. meningitidis are available in the art, and their nucleotide sequences are known.
  • nucleotide sequences encoding the chimeric fHBPs can be modified so as to optimize the codon usage to facilitate expression in a host cell of interest (e.g., E. coli, N. meningitidis, human (as in the case of a D ⁇ A-based vaccine), and the like). Methods for production of codon optimized sequences are known in the art.
  • Chimeric fHBPs can be produced by any suitable method, including recombinant and non-recombinant methods (e.g., chemical synthesis). Where the chimeric fHBP is produced using recombinant techniques, the methods can involve any suitable construct and any suitable host cell, which can be a prokaryotic or eukaryotic cell, usually a bacterial or yeast host cell, more usually a bacterial cell. Methods for introduction of genetic material into host cells include, for example, transformation, electroporation, conjugation, calcium phosphate methods and the like. The method for transfer can be selected so as to provide for stable expression of the introduced chimeric fHBP -encoding nucleic acid.
  • the chimeric fHBP -encoding nucleic acid can be provided as an inheritable episomal element (e.g., plasmid) or can be genomically integrated.
  • Suitable vectors for transferring chimeric fHBP-encoding nucleic acid can vary in composition. Integrative vectors can be conditionally replicative or suicide plasmids, bacteriophages, and the like.
  • the constructs can include various elements, including for example, promoters, selectable genetic markers (e.g., genes conferring resistance to antibiotics (for instance kanamycin, erythromycin, chloramphenicol, or gentamycin)), origin of replication (to promote replication in a host cell, e.g., a bacterial host cell), and the like.
  • selectable genetic markers e.g., genes conferring resistance to antibiotics (for instance kanamycin, erythromycin, chloramphenicol, or gentamycin)
  • origin of replication to promote replication in a host cell, e.g., a bacterial host cell
  • the choice of vector will depend upon a variety of factors such as the type of cell in which propagation is desired and the purpose of propagation.
  • vectors are useful for amplifying and making large amounts of the desired DNA sequence.
  • Other vectors are suitable for expression in cells in culture.
  • Still other vectors are suitable for transfer and expression in cells in a whole animal.
  • the choice of appropriate vector is well within the skill of the art. Many such vectors are available commercially.
  • the vector is an expression vector based on episomal plasmids containing selectable drug resistance markers and elements that provide for autonomous replication in different host cells (e.g., in both E. coli and N. meningitidis).
  • episomal plasmids containing selectable drug resistance markers and elements that provide for autonomous replication in different host cells (e.g., in both E. coli and N. meningitidis).
  • a "shuttle vector” is the plasmid pFPIO (Pagotto et al. Gene 2000 244:13- 19).
  • Constructs can be prepared by, for example, inserting a polynucleotide of interest into a construct backbone, typically by means of D ⁇ A ligase attachment to a cleaved restriction enzyme site in the vector.
  • the desired nucleotide sequence can be inserted by homologous recombination or site-specific recombination.
  • homologous recombination is accomplished by attaching regions of homology to the vector on the flanks of the desired nucleotide sequence, while site-specific recombination can be accomplished through use of sequences that facilitate site-specific recombination (e.g., cre-lox, att sites, etc.).
  • Nucleic acid containing such sequences can be added by, for example, ligation of oligonucleotides, or by polymerase chain reaction using primers comprising both the region of homology and a portion of the desired nucleotide sequence.
  • Vectors can provide for extrachromosomal maintenance in a host cell or can provide for integration into the host cell genome.
  • Vectors are amply described in numerous publications well known to those in the art, including, e.g., Short Protocols in Molecular Biology, (1999) F. Ausubel, et al., eds., Wiley & Sons.
  • Vectors may provide for expression of the nucleic acids encoding a chimeric fHBP, may provide for propagating the subject nucleic acids, or both.
  • Exemplary vectors that may be used include but are not limited to those derived from recombinant bacteriophage DNA, plasmid DNA or cosmid DNA.
  • plasmid vectors such as pBR322, pUC 19/18, pUC 118, 119 and the M13 mp series of vectors may be used.
  • pET21 is also an expression vector that may be used.
  • Bacteriophage vectors may include ⁇ gtlO, ⁇ gtl l, ⁇ gtl8-23, ⁇ ZAP/R and the EMBL series of bacteriophage vectors.
  • vectors that may be utilized include, but are not limited to, pJB8, pCV 103, pCV 107, pCV 108, pTM, pMCS, pNNL, pHSG274, COS202, COS203, pWE15, pWE16 and the charomid 9 series of vectors.
  • an expression cassette may be employed for expression of a chimeric fHBP of interest.
  • the present disclosure provides a recombinant expression vector comprising a subject nucleic acid.
  • the expression vector provides transcriptional and translational regulatory sequences, and may provide for inducible or constitutive expression, where the coding region is operably linked under the transcriptional control of the transcriptional initiation region, and a transcriptional and translational termination region. These control regions may be native to an fHBP from which the chimeric fHBP is derived, or may be derived from exogenous sources.
  • the transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. Promoters can be either constitutive or inducible, and can be a strong constitutive promoter (e.g., T7, and the like).
  • Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding proteins of interest.
  • a selectable marker operative in the expression host may be present to facilitate selection of cells containing the vector.
  • the expression construct may include additional elements.
  • the expression vector may have one or two replication systems, thus allowing it to be maintained in organisms, for example in mammalian or insect cells for expression and in a procaryotic host for cloning and amplification.
  • the expression construct may contain a selectable marker gene to allow the selection of transformed host cells. Selection genes are well known in the art and will vary with the host cell used.
  • chimeric fHBP of the present disclosure may comprise additional elements, such as a detectable label, e.g., a radioactive label, a fluorescent label, a biotin label, an immunologically detectable label (e.g., an HA tag, a poly-Histidine tag) and the like. Additional elements of chimeric fHBP can be provided to facilitate isolation (e.g., biotin tag, immunologically detectable tag) through various methods (e.g., affinity capture, etc.). Chimeric fHBP can optionally be immobilized on a support through covalent or non- covalent attachment.
  • a detectable label e.g., a radioactive label, a fluorescent label, a biotin label, an immunologically detectable label (e.g., an HA tag, a poly-Histidine tag) and the like.
  • Additional elements of chimeric fHBP can be provided to facilitate isolation (e.g., biotin tag, immunologically detectable tag) through
  • chimeric fHBP can be isolated from a lysate of cells genetically modified to express a chimeric fHBP, or from a synthetic reaction mix, by immunoaffinity purification, which generally involves contacting the sample with an anti- chimeric fHBP antibody (e.g., an anti-chimeric fHBP mAb, such as a JAR 5 mAb or other appropriate JAR mAb described herein), washing to remove non- specifically bound material, and eluting specifically bound chimeric fHBP.
  • Isolated chimeric fHBP can be further purified by dialysis and other methods normally employed in protein purification methods.
  • the chimeric fHBP can be isolated using metal chelate chromatography methods.
  • any of a number of suitable host cells can be used in the production of chimeric fHBP.
  • the chimeric fHBP described herein may be expressed in prokaryotes or eukaryotes, usually bacteria, more usually E. coli or Neisseria (e.g., N. meningitidis) in accordance with conventional techniques.
  • the present disclosure further provides a genetically modified host cell, which contains a nucleic acid encoding a chimeric fHBP.
  • Host cells for production (including large scale production) of a chimeric fHBP can be selected from any of a variety of available host cells.
  • Exemplary host cells for expression include those of a prokaryotic or eukaryotic unicellular organism, such as bacteria (e.g., Escherichia coli strains), yeast (e.g., S. cerevisiae, Pichia spp., and the like)., and may include host cells originally derived from a higher organism such as insects, vertebrates, particularly mammals, (e.g. CHO, HEK, and the like). Generally bacterial host cells and yeast are of particular interest for chimeric fHBP production.
  • bacteria e.g., Escherichia coli strains
  • yeast e.g., S. cerevisiae, Pichia spp., and the like
  • host cells originally derived from a higher organism such as insects, vertebrates, particularly mammals, (e.g. CHO, HEK, and the like).
  • Generally bacterial host cells and yeast are of particular interest for chimeric fHBP production.
  • Chimeric fHBPs can be prepared in substantially pure or substantially isolated form (i.e., substantially free from other ⁇ eisserial or host cell polypeptides) or substantially isolated form.
  • the chimeric fHBP is present in a composition that is enriched for the polypeptide relative to other components that may be present (e.g., other polypeptides or other host cell components).
  • Purified chimeric fHBP can be provided such that the polypeptide is present in a composition that is substantially free of other expressed polypeptides, e.g., less than 90%, usually less than 60% and more usually less than 50% of the composition is made up of other expressed polypeptides.
  • a ⁇ eisserial host cell is genetically modified to express a chimeric fHBP.
  • Any of a variety of Neisseria spp. strains can be modified to produce a chimeric fHBP, and, optionally, which produce or can be modified to produce other antigens of interest, such as PorA, can be used in the methods disclosed herein.
  • Methods and vectors to provide for genetic modification of ⁇ eisserial strains and expression of a desired polypeptide are known in the art. Exemplary vectors and methods are provided in WO 02/09746 and O'Dwyer et al. Infect Immun 2004;72:6511-80. Strong promoters, particularly constitutive strong promoters are of particular interest. Exemplary promoters include the promoters of porA, porB, lbpB, tbpB, pi 10, hpuAB, lgtF, opa, pi 10, 1st, hpuAB. and rmp.
  • Pathogenic Neisseria spp. or strains derived from pathogenic Neisseria spp., particularly strains pathogenic for humans or derived from strains pathogenic or commensal for humans, are of particular interest for use in membrane vesicle production.
  • Exemplary Neisserial spp. include N. meningitidis, N. flavescens, N. gonorrhoeae, N. lactamica, N. polysaccharea, N. cinerea, N. mucosa, N. subflava, N. sicca, N. elongata, and the like.
  • meningitidis strains are of particular interest for genetic modification to express a chimeric fHBP and for use in vesicle production.
  • the strain used for vesicle production can be selected according to a number of different characteristics that may be desired.
  • the strain may be selected according to: a desired PorA type (a "serosubtype", as described above), capsular group, serotype, and the like; decreased capsular polysaccharide production; and the like.
  • the production strain can produce any desired PorA polypeptide, and may express one or more PorA polypeptides (either naturally or due to genetic engineering).
  • Exemplary strains includes those that produce a PorA polypeptide which confers a serosubtype of Pl.7,16; Pl.19,15; Pl.7,1; Pl.5,2; P1.22a,14; Pl.14 ; Pl.5, 10; Pl.7,4; Pl.12,13; as well as variants of such PorA polypeptides which may or may not retain reactivity with conventional serologic reagents used in serosubtyping.
  • PorA polypeptides characterized according to PorA variable region (VR) typing (see, e.g., Russell et al.
  • the production strain can be a capsule deficient strain.
  • Capsule deficient strains can provide vesicle-based vaccines that provide for a reduced risk of eliciting a significant autoantibody response in a subject to whom the vaccine is administered (e.g., due to production of antibodies that cross-react with sialic acid on host cell surfaces).
  • "Capsule deficient” or “deficient in capsular polysaccharide” as used herein refers to a level of capsular polysaccharide on the bacterial surface that is lower than that of a naturally-occurring strain or, where the strain is genetically modified, is lower than that of a parental strain from which the capsule deficient strain is derived.
  • a capsule deficient strain includes strains that are decreased in surface capsular polysaccharide production by at least 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, 90% or more, and includes strains in which capsular polysaccharide is not detectable on the bacterial surface (e.g., by whole cell ELISA using an anti-capsular polysaccharide antibody).
  • Capsule deficient strains include those that are capsule deficient due to a naturally-occurring or recombinantly-generated genetic modification.
  • Naturally-occurring capsule deficient strains see, e.g., Dolan-Livengood et al. J. Infect. Dis. (2003) 187(10): 1616-28), as well as methods of identifying and/or generating capsule-deficient strains (see, e.g., Fisseha et al. (2005) Infect. Immun. 73(7):4070-4080; Stephens et al. (1991) Infect Immun 59(l l):4097-102; Frosch et al. (1990) MoI Microbiol. 1990 4(7): 1215-1218) are known in the art.
  • Modification of a Neisserial host cell to provide for decreased production of capsular polysaccharide may include modification of one or more genes involved in capsule synthesis, where the modification provides for, for example, decreased levels of capsular polysaccharide relative to a parent cell prior to modification.
  • Such genetic modifications can include changes in nucleotide and/or amino acid sequences in one or more capsule biosynthesis genes rendering the strain capsule deficient (e.g., due to one or more insertions, deletions, substitutions, and the like in one or more capsule biosynthesis genes).
  • Capsule deficient strains can lack or be non- functional for one or more capsule genes.
  • Of particular interest are strains that are deficient in sialic acid biosynthesis.
  • strains having a defect in sialic acid biosynthesis can be defective in any of a number of different genes in the sialic acid biosynthetic pathway.
  • strains that are defective in a gene product encoded by the N-acetylglucosamine- 6-phosphate 2-epimerase gene known as synX AAF40537.1 or siaA AAA20475, with strains having this gene inactivated being of especial interest.
  • a capsule deficient strain is generated by disrupting production of a functional synX gene product (see, e.g., Swartley et al. (1994) J Bacterid. 176(5): 1530-4).
  • Capsule-deficient strains can also be generated from naturally-occurring strains using non-recombinant techniques, e.g., by use of bactericidal anti-capsular antibodies to select for strains that reduced in capsular polysaccharide.
  • the strains can be selected so as to differ in one or more strain characteristics, e.g., to provide for vesicles that differ in the chimeric fHBP used, PorA, and the like.
  • the antigenic compositions contemplated by the present disclosure generally include vesicles prepared from Neisserial cells that express a chimeric fHBP. As referred to herein "vesicles” is meant to encompass outer membrane vesicles as well as microvesicles (which are also referred to as blebs).
  • the antigenic composition comprises outer membrane vesicles (OMV) prepared from the outer membrane of a cultured strain of Neisseria meningitidis spp. genetically modified to express a chimeric fHBP.
  • OMVs may be obtained from Neisseria meningitidis grown in broth or solid medium culture, preferably by separating the bacterial cells from the culture medium (e.g. by filtration or by a low-speed centrifugation that pellets the cells, or the like), lysing the cells (e.g. by addition of detergent, osmotic shock, sonication, cavitation, homogenization, or the like) and separating an outer membrane fraction from cytoplasmic molecules (e.g.
  • outer membrane fractions may be used to produce OMVs.
  • the antigenic composition comprises microvesicles
  • MV MV
  • blebs containing chimeric fHBP, where the MV or blebs are released during culture of a Neisseria meningitidis strain genetically modified to express a chimeric fHBP.
  • MVs may be obtained by culturing a strain of Neisseria meningitidis in broth culture medium, separating whole cells from the broth culture medium (e.g. by filtration, or by a low-speed centrifugation that pellets only the cells and not the smaller blebs, or the like), and then collecting the MVs that are present in the cell-free culture medium (e.g.
  • Strains for use in production of MVs can generally be selected on the basis of the amount of blebs produced in culture (e.g., bacteria can be cultured in a reasonable number to provide for production of blebs suitable for isolation and administration in the methods described herein).
  • An exemplary strain that produces high levels of blebs is described in PCT Publication No. WO 01/34642.
  • strains for use in MV production may also be selected on the basis of NspA production, where strains that produce higher levels of NspA may be of particular interest (for examples of N.
  • meningitidis strains having different ⁇ spA production levels see, e.g., Moe et al. (1999 Infect. Immun. 67: 5664).
  • Other strains of interest for use in production of blebs include strains having an inactivated G ⁇ 33 gene, which encodes a lipoprotein required for cell separation, membrane architecture and virulence (see, e.g., Adu-Bobie et al. Infect Immun. 2004;72:1914-1919).
  • the antigenic compositions of the present disclosure can comprise vesicles from one strain, or from 2, 3, 4, 5 or more strains, which strains may be homologous or heterologous, usually heterologous, to one another.
  • the strains may be homologous or heterologous with respect to PorA.
  • the vesicles can be prepared from strains that express more than one chimeric fHBP (e.g., 1, 2, 3, or more chimeric fHBP) which may be composed of fHBP amino acid sequences from different variants (v.l, v.2, or v.3) or subvariants (e.g., a subvariant of v.l, v.2, or v.3).
  • the antigenic compositions can comprise a mixture of OMVs and MVs presenting the same or different chimeric fHBPs, where the chimeric fHBPs may optionally present epitopes from different combinations of fHBP variants and/or subvariants and where the OMVs and/or MVs may be from the same or different strains.
  • Vesicles from different strains can be administered as a mixture, or can be administered serially.
  • the vesicles are optionally treated to reduce endotoxin, e.g., to reduce toxicity following administration.
  • endotoxin can be accomplished by extraction with a suitable detergent (for example, BRIJ-96, sodium deoxycholate, sodium lauroylsarcosinate, Empigen BB, Triton X-IOO, TWEEN 20 (sorbitan monolaurate polyoxyethylene), TWEEN 80, at a concentration of 0.1-10%, preferably 0.5-2%, and SDS).
  • a suitable detergent for example, BRIJ-96, sodium deoxycholate, sodium lauroylsarcosinate, Empigen BB, Triton X-IOO, TWEEN 20 (sorbitan monolaurate polyoxyethylene), TWEEN 80, at a concentration of 0.1-10%, preferably 0.5-2%, and SDS.
  • a detergent for example, BRIJ-96, sodium deoxycholate, sodium lauroylsarcosinate, Empigen BB, Triton X-IOO, TWEEN 20 (sorbitan monolaurate polyoxyethylene), TWEEN 80, at
  • the vesicles of the antigenic compositions are prepared without detergent, e.g., without use of deoxycholate.
  • detergent treatment is useful to remove endotoxin activity, it may deplete the native fHBP lipoprotein and/or chimeric fHBP (including lipidated chimeric fHBP) by extraction during vesicle production.
  • it may be particularly desirable to decrease endotoxin activity using technology that does not require a detergent.
  • strains that are relatively low producers of endotoxin lipopolysaccharide, LPS
  • the vesicles can be prepared from Neisseria mutants in which lipooligosaccharide or other antigens that may be undesirable in a vaccine (e.g. Rmp) is reduced or eliminated.
  • Vesicles can be prepared from N. meningitidis strains that contain genetic modifications that result in decreased or no detectable toxic activity of lipid A.
  • such strain can be genetically modified in lipid A biosynthesis (Steeghs et al. Infect Immun 1999;67:4988-93; van der Ley et al. Infect Immun 2001;69:5981-90; Steeghs et al. J Endotoxin Res 2004; 10: 113-9; Fissha et al, Infect Immun 73:4070,.2005).
  • the immunogenic compositions may be detoxified by modification of LPS, such as downregulation and/or inactivation of the enzymes encoded by lpxLl or lpxL2, respectively.
  • LPS low-power polypeptide
  • Production of a penta- acylated lipid A made in lpxLl mutants indicates that the enzyme encoded by lpxLl adds the C12 to the ⁇ -linked 3-OH C14 at the 2' position of Glc ⁇ II.
  • the major lipid A species found in lpxL2 mutants is tetra-acylated, indicating the enzyme encoded by lpxL2 adds the other C12, i.e., to the ⁇ -linked 3-OH C14 at the 2 position of Glc ⁇ I.
  • meningitidis strains genetically modified so as to provide for decreased or no detectable functional LpxLl -encoded protein.
  • Such vesicles provide for reduced toxicity as compared to N. meningitidis strains that are wild-type for LPS production, while retaining immunogenicity of chimeric fHBP.
  • LPS toxic activity can also be altered by introducing mutations in genes/loci involved in polymyxin B resistance (such resistance has been correlated with addition of aminoarabinose on the 4' phosphate of lipid A).
  • genes/loci could be pmrE that encodes a UDP-glucose dehydrogenase, or a region of antimicrobial peptide-resistance genes common to many enterobacteriaciae which could be involved in aminoarabinose synthesis and transfer.
  • the gene pmrF that is present in this region encodes a dolicol-phosphate manosyl transferase (Gunn J. S., Kheng, B. L., Krueger J., Kim K., Guo L., hackett M., Miller S. I. 1998. MoI. Microbiol. 27: 1171-1182).
  • PhoP-PhoQ regulatory system which is a phospho-relay two component regulatory system (e.g., PhoP constitutive phenotype, PhoPc), or low Mg++ environmental or culture conditions (that activate the PhoP-PhoQ regulatory system) lead to the addition of aminoarabinose on the 4' -phosphate and 2-hydroxymyristate replacing myristate (hydroxy lation of myristate).
  • This modified lipid A displays reduced ability to stimulate E-selectin expression by human endothelial cells and TNF secretion from human monocytes.
  • Polymyxin B resistant strains are also suitable for use, as such strains have been shown to have reduced LPS toxicity (see, e.g., van der Ley et al. 1994. In: Proceedings of the ninth international pathogenic Neisseria conference. The Guildhall, Winchester, England).
  • synthetic peptides that mimic the binding activity of polymyxin B may be added to the antigenic compositions to reduce LPS toxic activity (see, e.g., Rustici et al. 1993, Science 259:361-365; Porro et al. Prog Clin Biol Res. 1998;397:315-25).
  • Endotoxin can also be reduced through selection of culture conditions. For example, culturing the strain in a growth medium containing 0.1 mg-100 mg of aminoarabinose per liter medium provides for reduced lipid toxicity (see, e.g., WO 02/097646).
  • Antigen composition is used herein as a matter of convenience to refer generically to compositions comprising a chimeric fHBP as disclosed herein, which chimeric fHBP may be optionally conjugated to further enhance immunogenicity.
  • Compositions useful for eliciting antibodies, particularly antibodies against Neisseria meningitidis group B (NmB), in a human are specifically contemplated by the present disclosure.
  • Antigenic compositions can contain 2 or more different chimeric fHBPs, where the chimeric fHBPs may present epitopes from different combinations of fHBP variants and/or subvariants.
  • Antigenic compositions generally comprise an immunologically effective amount of chimeric fHBP, and may further include other compatible components, as needed.
  • immunologically effective amount is meant that the administration of that amount to an individual, either in a single dose, as part of a series of the same or different antigenic compositions, is effective to elicit an antibody response effective for treatment or prevention of a symptom of, or disease caused by, for example, infection by Neisseria, particularly N. meningitidis, more particularly Group B N. meningitidis.
  • This amount varies depending upon the health and physical condition of the individual to be treated, age, the capacity of the individual's immune system to produce antibodies, the degree of protection desired, the formulation of the vaccine, the treating clinician's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
  • Dosage regimen may be a single dose schedule or a multiple dose schedule
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of the antigenic compositions of the present invention in an amount sufficient to produce the desired effect, which compositions are provided in association with a pharmaceutically acceptable excipient (e.g., pharmaceutically acceptable diluent, carrier or vehicle).
  • a pharmaceutically acceptable excipient e.g., pharmaceutically acceptable diluent, carrier or vehicle.
  • the antigenic composition may be administered in conjunction with other immunoregulatory agents.
  • Antigenic compositions can be provided in a pharmaceutically acceptable excipient, which can be a solution such as a sterile aqueous solution, often a saline solution, or they can be provided in powder form. Such excipients can be substantially inert, if desired.
  • the antigenic compositions can further comprise an adjuvant.
  • Suitable adjuvants that can be used in humans include, but are not necessarily limited to, alum, aluminum phosphate, aluminum hydroxide, MF59 (4.3% w/v squalene, 0.5% w/v Tween 80TM, 0.5% w/v Span 85), CpG-containing nucleic acid (where the cytosine is unmethylated), QS21, MPL, 3DMPL, extracts from Aquilla, ISCOMS, LT/CT mutants, poly(D,L-lactide-co-glycolide) (PLG) microparticles, Quil A, interleukins, and the like.
  • alum aluminum phosphate, aluminum hydroxide
  • MF59 4.3% w/v squalene, 0.5% w/v Tween 80TM, 0.5% w/v Span 85
  • CpG-containing nucleic acid where the cytosine is unmethylated
  • QS21, MPL, 3DMPL extracts from Aqui
  • thr-MDP ⁇ -acetyl-muramyl-L-threonyl-D-isoglutamine
  • CGP 11637 referred to as nor- MDP
  • nor- MDP ⁇ -acetyl-nor-muramyl-L-alanyl-D-isoglutamine
  • CGP 19835A referred to as MTP-PE
  • RIBI RIBI
  • an adjuvant may be determined by measuring the amount of antibodies directed against the immunogenic antigen or antigenic epitope thereof.
  • Further exemplary adjuvants to enhance effectiveness of the composition include, but are not limited to: (1) oil-in- water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59TM (WO 90/14837; Chapter 10 in Vaccine design: the subunit and adjuvant approach, eds.
  • Span 85 (optionally containing MTP-PE) formulated into submicron particles using a microfluidizer, (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic -blocked polymer L121, and thr-MDP either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) RIBI TM adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components such as monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox TM); (2) saponin adjuvants, such as QS21 or Stimulon TM
  • IL-I interferons
  • M-CSF macrophage colony stimulating factor
  • TNF tumor necrosis factor
  • MPL monophosphoryl lipid A
  • 3dMPL 3-O-deacylated MPL
  • WO 00/56358 combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulsions e.g. EP-A-0835318, EP-A-0735898, EP-A-0761231; (7) oligonucleotides comprising CpG motifs (Krieg Vaccine 2000, 19, 618-622; Krieg Curr opin MoI Ther2001 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.
  • a polyoxyethylene ether or a polyoxyethylene ester e.g WO 99/52549
  • a polyoxyethylene sorbitan ester surfactant in combination with an octoxynol WO 01/21207
  • a polyoxyethylene alkyl ether or ester surfactant in combination with at least one additional non-ionic surfactant such as an octoxynol (WO 01/21152);
  • a saponin and an immunostimulatory oligonucleotide e.g.
  • a CpG oligonucleotide (WO 00/62800); (11) an immunostimulant and a particle of metal salt e.g WO 00/23105; (12) a saponin and an oil-in- water emulsion e.g. WO 99/11241; (13) a saponin (e.g QS21)+3dMPL+IM2 (optionally+a sterol) e.g WO 98/57659; (14) other substances that act as immunostimulating agents to enhance the efficacy of the composition.
  • Muramyl peptides include N-acetyl-muramyl-L- threonyl-D-isoglutamine (thr-MDP), N-25 acetyl-normuramyl-L-alanyl-D-isoglutamine (nor- MDP), N-acetylmuramyl-L-alanyl-D-isoglutarninyl-L-alanine-2-(l'-2'-dipalmitoyl— sn- glycero-3-hydroxyphosphoryloxy)-ethylamine MTP-PE), etc.
  • Adjuvants suitable for administration to a human are of particular interest.
  • the antigen compositions may comprise other components, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium, carbonate, and the like.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
  • the concentration of chimeric fHBP in a formulation can vary widely (e.g., from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight) and will usually be selected primarily based on fluid volumes, viscosities, and patient-based factors in accordance with the particular mode of administration selected and the patient's needs.
  • Chimeric fHBP-containing formulations can be provided in the form of a solution, suspension, tablet, pill, capsule, powder, gel, cream, lotion, ointment, aerosol or the like. It is recognized that oral administration can require protection of the compositions from digestion. This is typically accomplished either by association of the composition with an agent that renders it resistant to acidic and enzymatic hydrolysis or by packaging the composition in an appropriately resistant carrier. Means of protecting from digestion are well known in the art. [00182] Chimeric fHBP-containing formulations can also provided so as to enhance serum half-life of chimeric fHBP following administration.
  • the chimeric fHBP may be provided in a liposome formulation, prepared as a colloid, or other conventional techniques for extending serum half-life.
  • a liposome formulation prepared as a colloid, or other conventional techniques for extending serum half-life.
  • a variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728 and 4,837,028.
  • the preparations may also be provided in controlled release or slow-release forms.
  • the chimeric fHBP-containing antigenic compositions are generally administered to a human subject that is at risk of acquiring a Neisserial disease so as to prevent or at least partially arrest the development of disease and its complications.
  • An amount adequate to accomplish this is defined as a "therapeutically effective dose.” Amounts effective for therapeutic use will depend on, e.g., the antigenic composition, the manner of administration, the weight and general state of health of the patient, and the judgment of the prescribing physician.
  • Single or multiple doses of the antigenic compositions may be administered depending on the dosage and frequency required and tolerated by the patient, and route of administration.
  • the chimeric fHBP-containing antigenic compositions are generally administered in an amount effective to elicit an immune response, particularly a humoral immune response, in the host.
  • amounts for immunization will vary, and can generally range from about 1 ⁇ g to 100 ⁇ g per 70 kg patient, usually 5 ⁇ g to 50 ⁇ g/70 kg.
  • Substantially higher dosages e.g. 10 mg to 100 mg or more
  • the initial administration can be followed by booster immunization of the same of different chimeric fHBP-containing antigenic composition.
  • vaccination involves at least one booster, more usually two boosters.
  • immunization can be accomplished by administration by any suitable route, including administration of the composition orally, nasally, nasopharyngeally, parenterally, enterically, gastrically, topically, transdermally, subcutaneously, intramuscularly, in tablet, solid, powdered, liquid, aerosol form, locally or systemically, with or without added excipients.
  • parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa. (1980).
  • the antigenic compositions can be administered to a human subject that is immunologically naive with respect to Neisseria meningitidis.
  • the subject is a human child about five years or younger, and preferably about two years old or younger, and the antigenic compositions are administered at any one or more of the following times: two weeks, one month, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months, or one year or 15, 18, or 21 months after birth, or at 2, 3, 4, or 5 years of age.
  • PCR and cloned into pGEM-T-Easy (Promega).
  • the resulting plasmids were treated with the restriction enzymes Ndel and Xhol and the approximately 800 base-pair fragments containing the fHBP coding sequences were ligated into pET21b ( ⁇ ovagen) cut with the same two enzymes.
  • the plasmid clones were confirmed by DN A sequence determination of PCR products obtained by the amplification of the plasmid with primers specific for the T7 promotor and terminator regions.
  • the plasmids encoded the full-length fHBP proteins except for the amino-terminal 19 amino acid signal sequence and 7 presumably flexible N-terminal residues, and included a C-terminal hexa-histidine (HiS 6 ) tag originating from the pET21b plasmid.
  • HisS 6 C-terminal hexa-histidine
  • fHBP Chimera I lacking a signal sequence was constructed by PCR amplification of the region encoding residues 8-135 from genomic DNA from strain MC58 (fHBP v.l) and that encoding residues 136-255 from strain 8047 (v.2).
  • the two fragments, including an overlapping region of 48 base pairs centered around amino acids 136-139, were assembled by PCR amplification using external, gene-specific primers containing the Ndel and Xhol restriction sites.
  • the chimeric gene was cloned into pET21b as described for wild- type genes above.
  • Site-specific mutagenesis was used to test predictions of amino acid residues involved in anti-fHBP mAb epitopes and to create Chimera II. Mutagenesis was performed using the QuikChange II kit (Stratagene) using 10 ng of plasmid template and the manufacturer's protocols. For testing residues putatively involved in mAb epitopes, mutagenesis reactions were performed on pET21 -based plasmids encoding wild-type fHBP genes from various strains. A residue in a reactive sequence was changed to that naturally present in a non-reactive sequence or vice versa.
  • Chimera II site-specific mutagenesis was used to introduce the A174K substitution into the pET21 plasmid encoding Chimera I. Mutant fHBP genes were confirmed by DN A sequencing as described for the wild-type plasmids, above.
  • fHBP expression and purification Wild-type, mutant and chimeric fHBPs were expressed in Escherichia coli BL21(DE3) (Novagen). fHBP purifications were performed from 1 L cultures. Mid-exponential cultures (optical density at 600 nm of 0.5-0.6) were grown at 37 0 C, induced with 0.5 mM isopropylthiogalactoside for 3-4 h and the bacteria harvested by centrifugation. The cells were lysed by incubation with chicken egg white lysozyme (Sigma) and two freeze/thaw cycles.
  • Bacterial lysates were treated with DNase and RNase (Sigma) and protease inhibitors (Complete EDTA-free, Roche) and clarified by centrifugation at 13,000 x g.
  • Recombinant fHBPs were purified by nickel chelate chromatography using Ni-NTA agarose (Qiagen) and buffers recommended by the supplier. Fractions containing purified fHBP were pooled and dialyzed against PBS (Roche) containing 5% (w/v) sucrose and 0.01% NaN 3 , filter sterilized and stored at 4 0 C. [00195] mAb preparation.
  • hybridoma cell lines from spleens from CD-I mice immunized with recombinant fHBP variant group 2 (gene from strain 2996) or variant group 3 (gene from strain M1239) using methods previously described for preparation of hybridoma cell lines secreting mAbs against fHBP v.l protein (Welsch et al., J. Immunol. 2004).
  • mAbs were precipitated from tissue culture supernatants with 50% saturated (NH 4 ⁇ SO 4 and dialyzed against phosphate-buffered saline (PBS; Roche). IgG isotypes were determined using Clonotyping-AP reagents (Southern Biotech).
  • Binding of the mAbs to recombinant fHBP was measured by ELISA.
  • the wells of a microtiter plate (Immulon 2B; Thermo Electron Corp.) were coated with 1 ⁇ g/ml of recombinant fHBP in PBS and incubated overnight at 4 0 C.
  • the plates were blocked with PBS containing 0.1% Tween-20 (Sigma) (PBST) and 1% BSA (Sigma).
  • the primary antibodies were anti-fHBP mAbs (0.016 to 5 ⁇ g/ml) and the secondary antibody was rabbit anti-mouse IgG-alkaline phosphatase (Zymed; 1:5,000), each diluted in PBST. After 1 h at room temperature, alkaline phosphatase substrate (Sigma) was added and the absorbance at 405 nm was measured after 30 min. [00197] For competitive inhibition ELISAs, one mAb was held at a fixed concentration sufficient to obtain an OD at 405 nm of 1.0 determined by direct binding ELISA, as described above.
  • a second mAb of a different isotype was added together with the first mAb to the wells of a microtiter plate coated with the antigen as described above, at concentrations ranging from 0.4 to 50 ⁇ g/ml.
  • the secondary mAb was an isotype specific mouse anti-IgG- alkaline phosphatase conjugate.
  • the ELISA was developed as described above. [00198] Inhibition of binding of factor H. The ability of an anti-fHBP mAb to inhibit binding of fH to fHBP was measured by ELISA. Wells of a microtiter plate were coated with rfHBP as described above.
  • the membranes were washed, incubated with the different anti-fHBP mAbs (1 to 5 ⁇ g/ml) or, as a control for protein expression by the different clones, 0.02 ⁇ g/ml of Penta-His mAb (Qiagen).
  • the membranes were washed in PBST and incubated with a 1:10,000 dilution of a rabbit anti-mouse IgG-horseradish peroxidase conjugate (Zymed) and washed again.
  • the membranes were developed with a chemiluminescent substrate (ECL + ; GE Healthcare) and visualized on a Storm 840 imager (Molecular Dynamics).
  • Bactericidal activity Bactericidal activity.
  • Complement-mediated bactericidal activity was measured as described previously using washed, log-phase bacteria grown in Mueller-Hinton broth supplemented with 0.25 % glucose and 0.02 mM CMP-NANA to an OD 620 of 0.6.
  • the buffer was Dulbecco's phosphate buffered saline (Mediatech, Inc.) containing 0.9 mM CaCl 2 x 2 H 2 O, 0.5 mM MgCl 2 x 6 H 2 O and 1% (w/v) BSA.
  • the complement source was human serum from a healthy adult with no detectable intrinsic bactericidal activity.
  • bactericidal activity For synergism of mAb bactericidal activity, equal quantities of two mAbs ranging from 0.4 to 50 ug/ml (final concentration) were used.
  • the bactericidal activity (BC 50 ) of the mouse antiserum (or mAb combination) was defined by the dilution (or mAb concentration) that gave a 50% decrease in the number of CFU after 60 min incubation at 37 0 C as compared with the CFU at time 0 in the negative control reactions.
  • JAR 1 and JAR 3 are individually bactericidal when tested against certain strains expressing v.l fHBP.
  • the results summarized in Figure 18 are evidence that the epitopes recognized by each of the mAbs is surface-exposed on encapsulated meningococcal strains and capable of interacting with protective antibodies.
  • the protein was divided into three domains, designated A, B and C (Giuliani et all. Infect Immun 2005 ;73: 1151-60).
  • a domain is highly conserved across v.l and v.2 variant groups, whereas the B and C domains contain sequences that diverge among strains.
  • B and C domains contain sequences that diverge among strains.
  • the only known fHBP epitope interacting with a bactericidal mAb was located in the C domain at R204 (Giuliani et al. supra ). (Note that the convention of numbering amino acid residues beginning with the first residue after the signal sequence is adopted herein).
  • a chimeric protein vaccine composed of the B domain from a variant 3 strain (B 3 ) fused with the C domain of a variant 1 strain (Ci) failed to elicit protective bactericidal responses against strains with either variant 1 or 3 fHBP.
  • the inventors identified the location of an epitope defined by two anti-fHBP mAbs, JAR 3 and 5 (referred to herein as the "JAR3/5" epitope), in the Bi domain around residue G121. JAR 3 individually was known to be bactericidal with human complement against strains from sequence type (ST) complex 32 (Welsch et al.
  • J Immunol 2004;172:5606-15 and, both JAR 3 and JAR 5 when combined with other mAbs were broadly bactericidal against strains expressing sub variants of variant 1 fHBP (Welsch et al., supra ).
  • JAR 3 (Madico et al. J Immunol 2006;177:501-10) and JAR 5 also inhibited binding of factor H (fH) to the surface of encapsulated N. meningitidis strains.
  • fH factor H
  • the chimeric fHBP provided for a polypeptide that elicited broadly protective antibodies against v.2 or v.3 strains.
  • the animals developed serum bactericidal antibody responses against strains expressing fHBP v.l, v.2 or v.3 whereas as expected the serum bactericidal antibody responses of the control mice immunized with the wild-type recombinant fHBP v.l were nearly entirely restricted to strains expressing fHBP v.l , and those of the control mice immunized with the wild-type recombinant fHBP v.2 were restricted to v.2 or v.3 (which from previous studies were known to cross-react (Masignani 2007 J Exp Med, supra and Beernink 2007 J. Infect Dis supra ). The results provide proof of
  • a chimeric protein vaccine composed of the A 1 domain (which is highly conserved across variant 1 and 2), the N-terminal portion of the B 1 domain expressing the JAR 3/5 epitope fused with the distal terminal portion of the B 1 domain and C 1 domain elicited cross-protective bactericidal antibodies in immunized mice.
  • the JAR 11 epitope is expressed by about one-third of disease-producing N. meningitidis strains in the U.S. that express fHBP v.2 or v.3 (Beernink 2007 J. Infect Dis, supra). Approximately 50 percent of the JAR 11-negative strains with v.2 or v.3 fHBP express the JAR 32/35 epitope. Therefore, to increase coverage against these strains, the Chimera II vaccine was prepared, in which a single amino acid change, A174K, was introduced into the C domain that inactivated the epitope recognized by JAR 11 and introduced the epitope recognized by JAR 32/35.
  • JAR 32 is often associated with expression of JAR 33 (for example, strains 03S-0658, M1239 and SK104, Table in Figure 8A), while expression of JAR 11 is usually associated with expression of JAR 10 (see for example our strains 8047, MD1435 and MD1321, Figure 8A).
  • JAR 33 for example, strains 03S-0658, M1239 and SK104, Table in Figure 8A
  • JAR 11 is usually associated with expression of JAR 10 (see for example our strains 8047, MD1435 and MD1321, Figure 8A).
  • bactericidal activity of antibodies is enhanced when two antibodies bind their respective epitopes located at a distance of about 18 - 20 A. Stated differently, a greater bactericidal effect was observed when two antibodies bound epitopes located about 18-20 A apart on the chimeric fHBP compared to the bactericidal effect of these antibodies alone. In contrast, binding of two antibodies to epitopes positioned at a greater distance apart (e.g., > about 27 A) did not enhance bactericidal activity, which may be due to the reduced ability of these bound antibodies to provide for enhanced interaction with CIq of the complement cascade.
  • Binding of two antibodies to epitopes positioned ⁇ about 14 A apart also did not provide for enhanced bactericidal activity, which may be the result of steric hindrance of binding of one antibody by the other.
  • chimeric fHBP which contain epitopes that elicit antibodies that bind to epitopes within about 18 - 20 A apart can provide for further enhanced bactericidal antibody production. Examples of such epitope pairs are those epitopes bound by JAR 10 and JAR 11; and by JAR 33 and JAR 32/ 35 (JAR 32 and JAR 35 bind to the same (or overlapping) epitopes).
  • chimeric fHBP polypeptides as vaccines can be further enhanced by including epitopes that elicit antibodies that block fH binding.
  • epitopes that elicit antibodies that block fH binding.
  • JAR 13 v.2 epitope
  • JAR 11 v.2 epitope
  • JAR 32/35 v.3 epitope
  • binding of fHBP to fH is inhibited.
  • the presence of such fH-binding epitopes in the chimeric fHBP polypeptides can provide for production of antibodies that can facilitate protection through this pathway.
  • JAR 3 and JAR 5 were known to bind to v.l or subvariants of v.l fHBP expressed by strains MC58, 4243, M1390, and NZ98/254 but not to strain M6190 (Welsch et al. J Immunol 2004;172:5606-15).
  • fHBP expressed by M6190 had an arginine at position 121 (R121) whereas the fHBPs from the four reactive strains had glycine at position 121 (G121) (Welsch et al. J Immunol 2004; 172:5606- 15).
  • site-specific mutagenesis was used to change the glycine residue at position 121 in the fHBP sequence of strain MC58 to arginine.
  • Figures 30-32 provide supporting data for identification of residues involved in JAR mAb binding.
  • E. coli lysates containing plasmids expressing the respective wild-type and mutant fHBPs were analyzed by Western blot using the appropriate JAR mAb, as well as a control antibody to detect an epitope tag present on the fHBP (penta-His).
  • Figure 30 is a Western blot indicating residues involved in the
  • JAR 10 and JAR 33 epitopes in which E. coli lysates containing plasmids expressing the respective wild-type and mutant fHBPs were analyzed by Western blot with JAR 10 (Panel A) or Penta-His mAb (Panel B).
  • Figure 31 is a Western blot indicating a residue involved in the JAR 11, JAR 32 and JAR 35 epitopes, in which E.
  • Figure 32 is a Western blot indicating residue involved in the JAR 13 epitope, in which E. coli lysates containing plasmids expressing wild-type and mutant fHBPs were were probed with JAR 13 (Panel A) or Penta-His mAb (Panel B) and anti-mouse IgG-HRP secondary antibody.
  • the amino acid sequences of the v.2 protein (with fHBP from strain 8047 used as the reference v.2 amino acid sequence) and v.3 fHBP (with fHBP from strain M1239 used as the reference v.3 amino acid sequence) differ by -1 and +7 amino acid residues, respectively, from that of MC58.
  • a leucine residue (L) referred to using the numbering system herein as being at position 166 of the v.2 or v.3 fHBP sequence is actually at position 165 of the v.2 protein and at position 173 in the v.3 protein based on the actual amino acid sequence of these proteins, rather than on the numbering used herein based on the alignment of these sequences with v.1 fHBP of MC58.
  • FIG. 19 is a series of graphs showing the ability of representative JAR mAbs prepared against fHBP v.2 (8047), v.3 (MI239), or v.l (MC58) recombinant proteins to give concentration-dependent inhibition of binding of fH to rfHBP v.2 (Panel A), rfHBP v.3 (Panel B), or rfHBP v.l (Panel C) in an ELISA.
  • These data show that some of the contact residues defined by the JAR mAb epitopes are involved in binding to fH.
  • the epitopes defined by JAR 3 and JAR 5 are located within the B domain of variant 1 fHBP beginning approximately 19 amino acid residues N-terminal to the start of ⁇ -helix AH2, or approximately 15 amino acid residues N-terminal to the amino acid sequence of GEHT,.
  • a first chimeric fHBP (referred to herein as "Chimera I") was constructed by combining a portion of the gene encoding the A domain and the N-terminal portion of the B domain up to residue G136 from v.l fHBP of MC58 with the distal portion of the gene encoding the alpha-helix of the B domain and C domain of v.2 fHBP from strain 8047.
  • the residue at position G136 was used as a convenient crossover position (the point at which the chimeric sequence "shifted" from that of the v.l fHBP to that of the v.2 fHBP).
  • G136 is N-terminal the ⁇ -helix AH2 and begins a sequence of four highly conserved residues, GEHT, which are shown in a box in Figure 4.
  • the outer brackets show the region of the protein previously defined as the B domain (Giuliani et al. Infect Immun 2005 ;73: 1151- 60).
  • Chimera II was identical in amino acid sequence to Chimera I except for the introduction the A174K substitution (Bold and underlined K, Chimera II, Figure 4). It should be noted that the A domain is not shown in Figure 4, or in the NMR-based structure of Figure 5, but would be attached at the N-terminus labeled "N" in Figure 5.
  • Chimera I contains the JAR 11 epitope, including residue A174 ( Figure 5, Panel A).
  • Chimera II contains the JAR 32 and JAR 35 epitopes, including residue K174 ( Figure 5, Panel B).
  • the model of the fHBP chimeras was constructed based on the NMR structure of Cantini et al. J Biol Chem 2006;281:7220-7.
  • EXAMPLE 4 PURIFICATION AND CHARACTERIZATION OF MUTANT PROTEINS.
  • EXAMPLE 5 EPITOPE EXPRESSION BY CHIMERIC ANTIGENS.
  • ELISA was used to assess concentration-dependent binding of the anti-fHBP mAbs to the chimeric antigens isolated in Example 4.
  • JAR 1 which binds to a v.l epitope in the C domain (R204)
  • JAR 5 which is specific for an epitope on the B domain of fHBP v.l
  • JAR 4 which cross-reacts with an epitope that is not yet defined by expressed by v.l and v.2, showed identical respective concentration-dependent binding with the two chimeric proteins as compared with the respective wild-type v.l and/or v.2 proteins.
  • Figure 7, Panel B provides binding data for mAbs JAR 10, 11 and 13, which were from a mouse immunized with v.2 or fHBP. All three mAbs recognize epitopes on the C domain of fHBP v.2 of strain 8047 ( Figure 20), and they showed similar respective concentration-dependent binding with the Chimera I protein as they did with the wild-type rfHBP v.2 control protein expressed from the gene of strain 8047 (Fig. 7, Panel B).
  • JAR 11 did not bind to Chimera II, since this protein had lysine substituted for alanine at position 174 (A 174K), which eliminated the JAR 11 epitope and introduced the JAR 32 epitope (Panel C).
  • JAR 36 which cross-reacts with an epitope not yet defined but present on fHBP v.2 and v.3 fHBP bound to both of the chimeric proteins, and to the wild- type rfHBP v.2 control but not with the fHBP v.l control (Fig. 7, Panel C).
  • the proteins shown in Figure 6 were used to immunize mice according to the schedule in Table 1, below.
  • Four doses of vaccine (20 ⁇ g of protein) were administered IP with intervals of 2 weeks between doses. Mice were bled 2.5 weeks after dose 4.
  • CFA complete Freund's adjuvant
  • IFA incomplete Freund's adjuvant
  • Al(OH) 3 aluminum hydroxide.
  • CFA/IFA below indicates that the mice received an initial dose with CFA, then subsequent booster doses with IFA. Where Al(OH) 3 was used as the adjuvant, all doses were administered with aluminum hydroxide as the adjuvant.
  • Figure 9 summarizes the serum antibody responses of the mice that received the chimeric vaccines administered with FA when measured against strain H44/76 (variant 1) and four additional strains that express subvariants of v.l fHBP (i.e., fHBP proteins with relatively small amino acid differences (e.g., with greater than about 88%, and less than 97% amino acid sequence identity) between the sequence of the respective protein and that of v.1.1 fHBP of H44/76).
  • variant 1 strain H44/76
  • fHBP proteins with relatively small amino acid differences e.g., with greater than about 88%, and less than 97% amino acid sequence identity
  • mice immunized with the wild-type rfHBP v.l control protein had high responses to H44/76 (reciprocal GMT of nearly 10,000) and lower and variable responses against the other four test strains (ranging from a GMT of ⁇ l:10 against strain NZ98/254 to a GMT of >l:l,000 against SK084).
  • the sera from the mice immunized with the rfHBP v.2 control protein were either negative for bactericidal activity (bactericidal titers ⁇ l:10, four strains) or weakly positive (reciprocal GMT of 1:10; strain SK141).
  • mice immunized with either chimeric protein vaccine developed serum bactericidal antibodies against all four strains that were susceptible to bactericidal activity of sera from the control mice immunized with rfHBP v.1.
  • the respective reciprocal GMTs of the chimeric vaccine groups were ⁇ 1 log lower than those of mice given the control rfHBP v.l vaccine whereas the responses to the fifth strain, SK141, were as high or higher than those of the mice given the control rfHBP v.l vaccine.
  • Figure 10 summarizes the serum bactericidal antibody responses as measured against the six strains expressing fHBPs in the v.2 or v.3 variant groups. Three of the strains were JAR 11 positive (left panel) and three were JAR 32 positive (right panel). Sera from control mice immunized with rfHBP v.2 were bactericidal against all six strains whereas, with one exception (strain 03S-0658), the serum bactericidal titers of control mice immunized with rfHBP v.l were ⁇ l:10. The sera from the mice immunized with either chimeric vaccine were bactericidal against all six strains.
  • the respective titers were ⁇ 1 log lower than those of the control mice immunized with rfHBP v.2.
  • the titers elicited by one or both chimeric vaccines were similar to those of the mice given the positive control rfHBP v.2 vaccine.
  • the chimeric vaccines elicited bactericidal antibody responses against strains expressing fHBP from each of the three antigenic variant groups.
  • the Chimera I vaccine expressed the JAR 11 epitope while the Chimera II vaccine expressed the JAR 32 epitope (Fig. 1).
  • the respective responses of mice immunized with either chimeric vaccine when measured against strains expressing JAR 11- or JAR 32-positive fHBP were not significantly different from each other (Fig. 5).
  • FIG. 11 summarizes the serum bactericidal antibody responses of mice immunized with the chimeric vaccines when absorbed with aluminum hydroxide as measured against the panel of test strains with v.l fHBP.
  • the corresponding data for responses to test strains expressing v.2 or v.3 fHBP are shown in Figure 12.
  • the respective responses to the different vaccines absorbed with aluminum hydroxide paralleled those observed to the vaccines given with Freund's adjuvant although as expected the titers were somewhat lower with the aluminum hydroxide adjuvant.
  • JAR3/5 epitope which is common to virtually all v.l proteins but is not present in fHBP from v.2 or v.3 strains, is important for eliciting high bactericidal antibody titers to the variant 1 fHBP protein.
  • This discovery that the epitope recognized by JAR 3/5 plays an important role in eliciting v.1 fHBP bactericidal antibodies provides the basis for the rational design of further chimeric fHBP vaccines that can elicit bactericidal antibodies across strains expressing different fHBP protein variants, particularly against both v.l and v.2 fHBP-expressing strains.
  • the two chimeric vaccines described above included, from N-terminus to C-terminus:
  • a heterologous B domain composed of, from N-terminus to C-terminus, a) a contiguous amino acid sequence of an N-terminal portion of a B domain of a v.l fHBP containing the amino acid sequence defining the JAR 3/5 epitope, operably linked to b) a contiguous amino acid sequence defining the remainder of the B domain, which amino acid sequence is that of a B domain of a v.2 fHBP, where the v.2 fHBP amino acid sequence is present in the heterologous B domain following the amino acid sequence of GEHT; and
  • EXAMPLE 8 NATURAL OR SYNTHETIC POLYMORPHISMS OF v.l FHBP B DOMAINS
  • Figure 13 provides an alignment of fHBP v.l sequences with natural polymorphisms in the ⁇ -terminal portion of the B domain.
  • sequence conservation is shown below the alignment, with "*” denoting residues that are identical, “:"denoting residues that are conserved, and ".”denoting residues that are semi-conserved across the aligned sequences.
  • the positions of alpha-helices are shown above the alignment, with the residues implicated in the alpha helices indicated by italics.
  • the residue, G121 which is implicated in the JAR 3 and JAR 5 epitope, is shown in bold and underlining.
  • a lysine at position 122 also appears to be important in the JAR 3/5 epitope, since one strain that is negative for JAR 3 and JAR 5 binding, 03S-0408, has G121 but differs from JAR 3/5 reactive strains in having serine at position 122 (S122). (data not shown)
  • the amino acid sequence of GEHT that provides the point at which the B domain sequence of the chimera "switches” or "crosses over" to the amino acid sequence of a v.2 B domain in Chimera I and Chimera II (referred to herein as the "junction point" is shown in the box.
  • the alignment thus provides guidance as to the types of amino acid substitutions that can be introduced while maintaining JAR 3/5 epitope expression.
  • amino acid substitutions can be either naturally-occurring (i.e., natural polymorphisms) or can be introduced by synthetic means (e.g., recombinant polymorphisms).
  • the heterologous B domains of the chimeric fHBP polypeptides contemplated herein include those containing v.1 B domain sequences with natural and synthetic polymorphisms in the proximal B domain, including both natural and synthetic variants.
  • chimeric fHBPs contemplated herein generally include one or more epitopes of v.2 fHBP B and C domains, where the v.2 fHBP B domain epitopes are present at a location C-terminal to (i.e., distal to) the location of the JAR 3/5 epitope of v.l fHBPs, which is generally C-terminal to the alpha helix that follows the sequence of GEHT, which is shared between v.l and v.2 fHBPs.
  • Figure 14 provides an alignment of exemplary v.2 fHBP sequences of the distal portion of the B domain, and further provides exemplary v.2 fHBP sequences of the C domain, where the B domain is generally defined as residues 101 to 164, with numbering based on MC58 fHBP as a reference.
  • Figures 13 and 14 provide 7 exemplary amino acid sequences of the N-terminal region of the v.l B domain ( Figure 10) and 9 exemplary amino acid sequences for the distal portion of the B domain of v.2 fHBP and for the C domain of v.2 fHBP ( Figure 11).
  • Figures 13 and 14 provide for a least 63 different fHBP chimeras, simply by combining the amino acid sequences as provided. Further, the alignments provide guidance as to the amino acid residues that can be substituted.
  • these exemplary sequences provide guidance for use of amino acid sequences containing naturally-occurring polymorphisms, they also provide guidance for production of synthetically-generated variants (e.g., using recombinant methods).
  • additional point mutations may be introduced into the coding nucleic acid to provide for fHBP chimeras that express other desirable epitopes, such as the v.l epitope recognized by JAR 1/mAb 502 that is in the portion of the C domain (R204), or the JAR 11 epitope in Chimeras IV and V.
  • JARl/mAb 502 epitope can provide for improved cross-reactivity against v.l strains from the ST-32 lineage that nearly always express the JAR 1 epitope, while eliciting antibodies to the JAR 11 epitope can provide for improved titers against v.2 strains (see Koeberling et al., J Infect Dis 2008, 198:262-270, for a description of v.l.1).
  • JAR 32 is often associated with expression of JAR 33
  • JAR 11 is usually associated with expression of JAR 10 (see, for example, strain panel, Figure 8).
  • Table 2 and Figures 15 and 16 provide for additional hypothetical chimeric vaccines, designated Chimera III, Chimera IV and Chimera V.
  • Each of these chimeric fHBPs are generated using a strategy similar to that used to generate Chimera I to provide for a chimeric fHBP that elicits bactericidal anti-v.l and v.2 antibodies.
  • Chimera III contains the A domain and proximal B domain of subvariant v.l.10 encoded by the fHBP from NZ98/254, and uses the same distal
  • Chimera IV illustrated in Figure 15, includes the A and proximal B domains of Chimera I and Chimera II, but substitutes the distal B and C domains of v.2 from strain
  • V vaccine antigens as compared with the respective amino acid sequences of the A and proximal B domain of MC58 and distal B and C domains of 8047 used to prepare Chimera 1 are shown in Figure 16.
  • Chimeras III and V can provide for increased breadth of protection against certain strains expressing subvariants of v.l, such as NZ98/254, which is not covered by the Chimera I or II vaccines.
  • Chimeras IV and V can provide for extended protection against v.3 strains, which are poorly covered by Chimera I or Chimera II.
  • Table 2 provides a summary of the observed epitope expression for Chimera I and Chimera II, and further sets out the predicted epitope expression for proposed Chimeras III, IV, and V.
  • Further modifications of the chimeric fHBP include varying the residue within the heterologous B domain at which the v.l fHBP B domain sequence ends and the v.2 (or v.2) fHBP B domain sequence ends.
  • residue G136 was used as the position of the "crossover" between fHBP variants, the "crossover" residue can be any residue C-terminal of the amino acid sequence defining the JAR 3/JAR 5 epitope and a residue within M123-S136 can also be selected.
  • residue positions C-terminal to G136 (E137-D142), or residue positions adjacent or within ⁇ -helix AH2 (K143-A150, preceding the first beta-strand) can be suitable crossover residue positions.
  • crossover residue positions are contemplated herein, where the crossover residue may be positioned after G121 of the JAR 3/5 epitope (G121) through and including A 174 of the JAR 11 epitope, where the presence of the JAR 3/5 epitope can be assessed using immunoassay methods known in the art, (Note that numbering above is based on the amino acid sequence number of the fHBP reference sequence of MC58.)
  • Figure 33 shows a Western blot of samples of N. meningitidis expressing either wildtype (WT) or a chimeric fHBP (Chimera I (see Fig. 23)).
  • the Chimera I corresponded to a full-length fHBP and further included the signal sequence (but lacked any epitope tags, such as a Penta-His tag)
  • bacterial cells transformed with the plasmid containing the gene encoding Chimera I were positive for both anti-fHbp mAbs, whereas the cells from H44/76 transformed with the plasmid containing the gene encoding fHbp v.2 or WT H44/76 react only with JAR 3 (v.l) and the cells from 8047 react only with JAR 13 (v.2).
  • Hybridomas producing the JAR 4, JAR 5, JAR 11, and JAR 32 monoclonal antibodies were deposited under the terms of the Budapest Treaty with the American Type Culture Collection, 10801 University Boulevard., Manassas, Va. 20110-2209, USA (ATCC) on the date indicated in the table below, and were assigned the designations set out in the table below.
  • JAR 5 mAb specifically binds to an epitope that at least overlaps with the epitope specifically bound by JAR 3 mAb
  • JAR 32 mAb specifically binds to an epitope that at least overlaps with the epitope specifically bounds by JAR 35 mAb.

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Abstract

Chimeric fHBPs that can elicit antibodies that are bactericidal for different fHBP variant strains of N. meningitidis, and methods of use, are provided.

Description

CHIMERIC FACTOR H BINDING PROTEINS TFHBP) CONTAINING A HETEROLOGOUS B DOMAIN AND METHODS OF USE
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority benefit of U.S. provisional application serial no. 61/035,329, filed March 10, 2008 and U.S. provisional application serial no. 61/037,252, filed March 17, 2008, both of which are incorporated herein by reference in their entirety.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under Public Health
Service grant nos. ROl AI46464 and C06 RR 16226. The government has certain rights in this invention.
FIELD OF THE INVENTION
[0003] This invention relates to vaccines for diseases caused by Neisseria meningitidis.
BACKGROUND
[0004] Neisseria meningitidis is a Gram-negative bacterium which colonizes the human upper respiratory tract and is responsible for worldwide sporadic and cyclical epidemic outbreaks of, most notably, meningitis and sepsis. The attack and morbidity rates are highest in children under 2 years of age. Like other Gram-negative bacteria, Neisseria meningitidis typically possess a cytoplasmic membrane, a peptidoglycan layer, an outer membrane which together with the capsular polysaccharide constitute the bacterial wall, and pili, which project into the outside environment. Encapsulated strains of Neisseria meningitidis are a major cause of bacterial meningitis and septicemia in children and young adults. The prevalence and economic importance of invasive Neisseria meningitidis infections have driven the search for effective vaccines that can confer immunity across different strains, and particularly across genetically diverse group B strains with different serotypes or serosubtypes.
[0005] Factor H Binding Protein (fHBP, also referred to in the art as lipoprotein 2086
(Fletcher et al, Infect Immun 2004;72:2088-2100), Genome-derived Neisserial antigen (GNA) 1870 (Masignani et al. J Exp Med 2003;197:789-99) or "741") is an N. meningitidis protein which is expressed in the bacterium as a surface-exposed lipoprotein. Based on sequence analysis of 71 N. meningitidis strains representative of its genetic and geographic diversity, N. meningitidis strains have been sub-divided into three fHBP variant groups (referred to as variant 1 (v.l), variant 2 (v.2), and variant 3 (v.3)) based on amino acid sequence variability and immunologic cross-reactivity (Masignani et al. J Exp Med 2003; 197:789-99). Other workers (Fletcher et al, 2004) have subdivided the protein into two sub-families designated A (which includes v.2 and v.3 of Masignani) and B (v.l). Variant 1 strains account for about 60% of disease-producing group B isolates (Masignani et al. 2003, supra). Within each variant group, there is on the order of about 92% or greater conservation of amino acid sequence. Specifically, conservation within each variant group ranges between 89 and 100%, while between the variant groups (e.g., between v.l and v.2) the conservation can be as low as 59%. The protein is expressed by all known strains of N. meningitidis. [0006] Mice immunized with recombinant fHBP developed high serum bactericidal antibody responses against strains expressing fHBP proteins of the homologous variant group (Masignani et al. 2003, supra; Welsch et al. 2004, J Immunol. 172(9):5606-15.). Thus, antiserum prepared against fHBP v.l confers protection against N. meningitidis strains expressing fHBP v.l, but not against strains expressing fHBP v.2 or v.3. Similarly, antiserum prepared against fHBP v.2 protects against strains expressing v.2 (or v.3) but not v.l (Masignani et al. J Exp Med 2003, 197:789-99; Beernink et al. J Infect Dis 2007; 195: 1472- 9). For vaccine purposes, it would be desirable to have a single protein capable of eliciting cross-protective antibodies against fHBP from different variant groups. [0007] Chimeric proteins have been used for vaccine development in a variety of ways. For example, a first strategy employs a genetic or chemical linkage of an antigen to a known, but unrelated, immunogenic protein, such as the diphtheria, tetanus or pertussis toxoid proteins, or the cholera toxin B (CTB) domain, in order to enhance the magnitude of the antibody responses to the antigen of interest. A second strategy uses a genetic fusion of two antigens from the same organism, to enhance cross-protection against strains with antigenic diversity (Giuliani et al. Infect Immun 2005 73:1151-60). An example is the multivalent group B meningococcal recombinant protein vaccine, which contains a mixture of two fusion proteins: a first fusion protein of a GΝA2091 protein and a GNAl 870 (or "fHBP") protein, and a second fusion protein of a GNA2132 protein and a GNA1030 protein (Giuliani et al. Proc Natl Acad Sci U S A 2006, 103:10834-9). A third strategy has been to construct a fusion of different serologic variants ("serovars") of one antigen to induce cross-
9 protection against a strains with antigenic diversity. An example is a tetravalent OspC chimeric Lyme disease vaccine, which induced bactericidal antibody responses against spirochete strains expressing each of the OspC types that were incorporated into the construct (Earnhart et al. Vaccine 2007;25:466-80).
[0008] In the examples of chimeric vaccines described that were designed to broaden protective immune responses, the vaccines were composed of repeats of an individual domain with antigenic variability. The respective variants of the domain were expressed in tandem in one protein (i.e., the same domain from different strains, A1-A2-A3-A4, etc). In some cases, these recombinant tandem proteins can be convenient for manufacturing and quality control. However they also can be very large and subject to improper folding or degradation. [0009] One approach to avoiding the problem of large tandem fusion proteins is to design a single polypeptide that is composed of different domains of two antigenic variants e.g., by "swapping" different individual domains of an antigen, or even smaller regions such as individual epitopes from two different proteins, to form a chimeric protein that expresses antigenically unrelated epitopes specific for more than one strain (i.e., different domains from two different strains, A1-B2 or A2-B1, etc.).
[0010] This latter approach was undertaken with fHBP. First, in order to facilitate identification of bactericidal regions of fHBP, the protein was divided into three domains, designated A, B and C (Giuliani (2005) Infect. Immun. 73:1151-1160). The A domain is highly conserved across variant groups, whereas the B and C domains contain sequences that diverge among strains. Giuliani et al. identified an fHBP epitope interacting with a bactericidal mAb located in the C domain at R204 (Giuliani (2005) supra). However, a chimeric protein containing the B domain from a variant 3 strain (B3) fused with the C domain of a variant 1 strain (C1) failed to elicit protective bactericidal responses against strains with either v.l or v.2 fHBP.
[0011] Vaccines that exploit the ability of fHBP to elicit bactericidal antibody responses and that can elicit such antibodies that are effective against strains expressing different fHBP variants remain of interest.
SUMMARY
[0012] Chimeric fHBPs that can elicit antibodies that are bactericidal for different fHBP variant strains of N. meningitidis, and methods of use, are provided. BRIEF DESCRIPTION OF THE DRAWINGS
[0013] Figure 1 is a collection of results of Western blot analysis illustrating the amino acid residues involved in binding of monoclonal antibodies (mAbs) JAR 3 and JAR 5 to factor H binding protein (fHBP). Panel A, JAR 5; lane 1, molecular mass standard; lane 2, pET21b; lane 3, pET21-fHBP(MC58 wildtype); lane 4, pET21-fHBP(MC58)G121R; lane 5, pET21-fHBP(M6190 wildtype)R121; lane 6, pET21-fHBP(M6190)R121G. Panel B, JAR 3. C, Penta-His mAb. Panels B and C have the same lane assignments as panel A. [0014] Figure 2 is a set of graphs illustrating that binding of JAR 3 and JAR 5 mAbs to fHBP is competitive. Percent competitive inhibition of binding of anti-fHBP mAbs to fHBP by a second antibody as measured by ELISA. Each panel includes: rabbit polyclonal anti-fHBP antiserum; rabbit pre-immune serum; and a negative control mAb specific for an irrelevant capsular antigen (JW-C2, -A2 or -Al). Panel A, Inhibition of binding of JAR 3 by JAR 4 or JAR 5. Panel B, Inhibition of binding of JAR 5 by JAR 3 or JAR 4. Panel C, Inhibition of binding of JAR 4 by JAR 3 or JAR 5.
[0015] Figure 3 is a schematic illustrating positions of residues associated with the epitopes of the nine anti-fHBP mAbs ("JAR" mAbs) in the structural model based on previously reported NMR data (Cantini et al. "Solution structure of the immunodominant domain of protective antigen GNA1870 of Neisseria meningitidis ." J Biol Chem 2006; 281:7220-7). Coordinates from the solution structure of the B and C domains of fHBP v.l from strain MC58 were used to construct the model. Note that the positions of amino acid residues involved in the epitopes for antibodies raised against the fHBP v.2 and v.3 proteins are shown on the model, even though these antibodies do not bind to the v.1 protein from strain MC58. It should also be noted that numbering of amino residues is based on the mature protein sequence of fHBP (i.e. lacking the signal sequence) from strain MC58. Because the amino acid sequences of the variant 2 (v.2) fHBP protein (from strain 8047) and variant 3 (v.3) fHBP (from strain M1239) differ by -1 and +7 amino acid residues, respectively, from that of MC58, the numbering used to refer to residues for v.2 and v.3 fHBP proteins differs from numbering based on the actual amino acid sequences of these proteins. Thus, for example, reference to a leucine residue (L) at position 166 of the v.2 or v.3 fHBP sequence in Figure 3, refers to the residue at position 165 of the v.2 protein and at position 173 in the v.3 protein. For further clarification, see Figure 4 for alignment. Details of the reactive and non-reactive residues are provided herein. The residue shown for mAb 502 is from a previously reported study (Giuliani et al., 2005 Infect Immun 73:1151-60). The numbering is based on amino acid sequence of MC58 v.l fHBP lacking the signal sequence (Masignani et al., 2003 J Exp Med 197:789-99).
[0016] Figure 4 is a schematic providing an alignment of wild-type and chimeric fHBP amino acid sequences (alignment performed using ClustalW). The deduced amino acid sequences of fHBP v.l from strain MC58 (bottom) and v.2 strain 8047 (top) are shown, along with the two chimeric sequences (middle, Chimera I and Chimera II). Numbering for all four proteins is based on the native, mature v.l protein from MC58 (i.e., without the signal sequence). The recombinant fHBP protein as expressed in E. coli lacks both the signal sequence and seven presumably flexible residues (CSSGGGG). An N-terminal methionine was added to each sequence shown to facilitate expression in E. coli (not shown). A C- terminal sequence LEHHHHHH was added to each sequence shown to facilitate isolation (not shown). The identities of the chimeras with the respective wild-type sequences are shown with symbols above and below the alignment (* = identical; : = conserved; .= semi- conserved). The position of the amino acid sequence of GEHT at residues 136-139 in the C- terminal portion of the B domain of 8047 following the junction point is indicated in a box. The outer brackets, which encompass residues 101 to 164, show the region of the protein defined as the B domain (Giuliani et al. "The region comprising amino acids 100 to 255 of Neisseria meningitidis lipoprotein GNA 1870 elicits bactericidal antibodies." Infect Immun 2005; 73:1151-60). With one exception, Chimera I and Chimera II have identical amino acid sequences. The exception is at residue 174 where alanine in Chimera I has been replaced by lysine in Chimera II. The position of the A174K substitution in Chimera II is shown in bold. Sequence alignment was performed with ClustalW.
[0017] Figure 5 is a schematic representation of chimeric fHBPs. The N-terminal portion of the B domain from the v.1 fHBP is on the right, and the C-terminal portion of the B domain encompassing the α-helix of the v.2 protein together with the C domain of the v. 2 fHBP is on the left. The junction point for these chimeras, exemplified by G136 is indicated with an arrow and accompanying text. Both chimeric proteins express the JAR 3 and JAR 5 epitopes expressed on the B domain of fHBP v.l and the JAR 10 epitope, which is on the C domain of subsets of strains expressing v.l, v.2 or v.3 fHBP. Chimera I contains the JAR 11 epitope, including residue A174 (Panel A), which is expressed on the C domains of a subset of strains expressing fHBP v.2 or v.3. Chimera II contains the JAR 32 /JAR 35 epitopes, including residue K174, which are expressed on the C domains of subsets of strains expressing fHBP v.2 or v.3. A domains are not shown in these representations. The model was constructed based on the NMR structure of Cantini et al. (2006) J Biol Chem 281:7220-7.
[0018] Figure 6 provides the results of SDS-PAGE analysis of purified wild-type and mutant fHBPs. Proteins were expressed from pET21-based plasmids in E. coli BL21(DE3) as C-terminal hexa-histidine fusions and purified by metal chelate chromatography. Proteins were dialyzed against Ix PBS, 5% sucrose, 1 mM DTT and filter sterilized. Proteins (5 μg each) were separated on a 4-12% polyacrylamide gel and stained with Coomassie blue. Lane 1, mass standard; 2, fHBP v.l (MC58); 3, fHBP v.2 (8047); 4, fHBP Chimera I; 5, fHBP Chimera II.
[0019] Figure 7 is a set of graphs illustrating binding of individual anti-fHBP mAbs to recombinant proteins. Panel A shows mAbs prepared against fHBP v.l; Panel B, fHBP v.2; Panel C, fHBP v.3. The symbols represent different antigens on the plate: open squares, fHBP v.l; open circles, fHBP v.2; open triangles, Chimera I; asterisks, Chimera II. [0020] Figure 8 A provides a table of strains used in the Examples, including those used to measure serum bactericidal antibody responses and description of the amino acid sequence identity compared with prototype fHBP v.l, v.2 and v.3 and JAR mAb binding of the respective fHBPs.
[0021] Figure 8B shows the amino acid identities of different domains of fHBP.
Comparisons are made for the A domain (residues 1-100), the B domain (residues 101-164) and the C domain (residues 165-255). Comparisons also are made for the B domain up to the junction point (101-135) and the B domain starting at the junction point (136-164). Numbering of amino acid residues is based on the mature protein (i.e. lacking the signal sequence) from strain MC58.
[0022] Figure 9 is a graph illustrating serum bactericidal antibody responses of mice immunized with chimeric recombinant proteins given with Freund's adjuvant as measured against N. meningitidis group B strains expressing fHBP in the antigenic v.l group. Strain H44/76 expresses fHBP v.1 identical to that of the fHBP v.1 control vaccine. The remaining strains express subvariants of fHBP v.l (See Table in Figure 8A, above). Values are presented as 1/GMT (Reciprocal (or inverse) geometric mean titer) with a 95% confidence interval.
[0023] Figure 10 is a graph illustrating serum bactericidal antibody responses of mice immunized with chimeric recombinant proteins given with Freund's adjuvant as measured against N. meningitidis group B expressing fHBP in the v.2 or v.3 antigenic groups. Strain
8047 expresses fHBP v.2 identical to that of control rfHBP v.2 vaccine. The remaining strains express subvariants of fHBP v.2 or v.3 (see Table in Figure 8A). Values are presented as 1/GMT with a 95% confidence interval. The data are stratified based on strains reacting with JAR 11 (left panel) or JAR 32 (right panel). The Chimera I and II vaccines are identical except that Chimera I has residue A174 and is JAR 11-positive and JAR 32-negative, whereas Chimera II has residue K174 and is JAR 11-negative and JAR 32-positive. See figure for bar symbols.
[0024] Figure 11 is a graph illustrating serum bactericidal antibody responses of mice immunized with chimeric recombinant proteins adsorbed to aluminum hydroxide as measured against N. meningitidis group B strains expressing fHBP in the antigenic v.l group. Strain H44/76 expresses fHBP v.l identical to that of the fHBP v.l control vaccine. The remaining strains express subvariants of fHBP v.l. Values are presented as 1/GMT (i.e., reciprocal (or inverse) geometric mean titer) with a 95% confidence interval. Bar symbols for each vaccine are as shown in Figure 10.
[0025] Figure 12 is a graph illustrating serum bactericidal antibody responses of mice immunized with chimeric recombinant proteins adsorbed to aluminum hydroxide as measured against N. meningitidis group B expressing fHBP in the v.2 or v.3 antigenic groups. Strain 8047 expresses fHBP v.2 identical to that of control rfHBP v.2 vaccine. The remaining strains express subvariants of fHBP v.2 or v.3 (see Table in Figure 8). Values are presented as 1/GMT with a 95% confidence interval. The data are stratified based on strains reacting with JAR 11 (left panel) or JAR 32 (right panel). The Chimera I and II vaccines are identical except that Chimera I is JAR 11-positive and JAR 32-negative, whereas Chimera II is JAR 11-negative and JAR 32-positive Bar symbols for each vaccine are as shown in Figure 10. [0026] Figure 13 is a schematic showing alignment of fHBP v.l amino acid sequences with natural polymorphisms in the Ν-terminal portion of the B domain. In the alternative nomenclature based on three dimensional structural data of the entire fHbp molecule, the sequence shown also comprises a C-terminal portion of the fHbpΝ domain and a small Ν-terminal portion of the fHbpC domain as indicated above the alignment. The sequence conservation is shown below the alignment (code as in Figure 4). The positions of α-helices are shown above the alignment. The position of the junction point in the chimeric proteins is shown in the box. Numbering is based on the mature protein (i.e. lacking the signal sequence) from strain MC58. Strains MC48, M4105, 4243, NZ98/254 are positive for JAR3/JAR 5 reactivity; strains M6190 and 03S-0408 are negative for JAR 3/5 reactivity, and strains NM452 and CDC1573 have not been tested. The residues G121 and K122, which are associated with JAR 3 and JAR 5 mAb epitopes, are shown in bold and underlined text. Note that although strain 03S-0408 has G121, it is negative for JAR 3/5 reactivity. This strain has three amino acid differences between positions 101 and 146 compared with amino acids of MC58: L109, V114 and S122. Since both L109 and V114 are present in reactive sequences, e.g. 4243 and M4105, lack of reactivity of 03S-0408 is likely attributable to the presence of serine at position 122 instead of lysine and, therefore in addition to G121, K122 also is associated with JAR3/5 reactivity.
[0027] Figure 14 is a schematic showing alignment of fHBP v.2 amino acid sequences with natural polymorphisms in the carboxyl-terminal portion of the B domain and the C domain, or alternatively, the complete fHbpC domain based on the three-dimensional structural nomenclature. The sequence conservation is shown below the alignment (code as in legend to Figure 4). The residues implicated in anti-fHBP mAb epitopes are designated with the number of the JAR mAb above the alignment: JAR 11 (alanine at residue position 174; A174); JAR 10 (lysine at residue position 180 and glutamate at position 192; K180 and E192); JAR 13 (serine residue at position 216; S216). Numbering in this figure is based on fHBP from strain MC58.
[0028] Figure 15 is a schematic illustrating additional exemplary chimeric vaccines
(Chimeras lib, III, IV, and V). Chimera lib can be made by introducing the Kl 80R substitution into Chimera II. Chimeras III and V can be made using portions of the A and B domains of strain NZ98/254 (subvariant v.1) with the distal portion of the B domain and C domain of v.2 strain 8047 (Chimera III) or of subvariant v.2 strain RM1090 (Chimera V). Chimera IV uses the A and proximal B domains of MC58 with the distal B and C domains of RM 1090.
[0029] Figure 16 is a schematic showing an alignment of amino acid sequences of further exemplary chimeric fHBPs (Chimera III, IV and V) in the region of the crossover position, which is indicated by the box (residues GEHT). The residues, G121 and K122, implicated in the JAR 3 and JAR 5 epitopes are shown in bold and underlining. [0030] Figure 17 provides a table summarizing cross-reactivity of the different JAR mAbs, their respective Ig isotypes and ability to inhibit binding of human fH. [0031] Figure 18 is a table listing human complement- mediated bactericidal activity of each of the JAR mAbs when tested individually or in combination with a second anti- fHBP mAb.
[0032] Figure 19 is a series of graphs showing the ability of representative JAR mAbs prepared against fHBP v.2 or v.3 proteins to give concentration-dependent inhibition of binding of fH to rfHBP in an ELISA. Panel A, Inhibition of binding of fH to rfHBP v.2. Panel B, Inhibition of binding of fH to rfHBP v.3. Respective v.2 and v.3 recombinant proteins are those encoded by the fHBP genes of strains 8047 and M1239. Panel C, Inhibition of binding of fH to rfHBP v.1.
[0033] Figure 20 is a table listing certain properties of respective pairs of JAR mAbs with or without synergistic complement-mediated bactericidal antibody, including the positions of amino acid residues involved in the epitopes, distances between them, inhibition of fH binding and isotype of each mAb.
[0034] Figure 21 provides the amino acid sequence of variant 1 (v.l) factor H binding protein (fHBP) of MC58, with the A, B and C domains indicated. Positions of the structural domains, fHbpN and fHbpC, are also shown. Glutamine 101 (Q) and glycine 164 (G) indicated by upward arrows define the A/B and B/C domain borders, respectively, as defined by Giuliani et al., Infect. Immun, 2005 73:1151-60. The upward arrow at glycine 136 designates the boundary between the fHbpN and the fHbpC domains, as defined by Cantini et al., J. Biol. Chem. 2009.
[0035] Figure 22 shows the amino- (N-) and carboxyl- (C-) terminal portions of the B and C domains, which are defined with respect to the conserved amino acid sequence of GEHT. The amino acid sequences that can define a JAR 3/5 epitope are positioned N- terminal to the second alpha helix; the amino acid sequence that can define the JAR 11/32/35 epitopes are positioned C-terminal to the second alpha helix. Alpha-helix (AH) 2 is indicated. [0036] Figure 23 is a schematic showing Chimera I (MC58/8047) nucleotide and protein sequences. The sequence before the junction (cross-over) point is shown in lower case and the sequence following the junction point is shown in upper case. Lines of fifty residues are shown. Only the Neisserial sequences are shown; E. coli expression constructs contained an N-terminal Methionine and C-terminal hexa-histidine tag (LEHHHHHH). [0037] Figure 24 is a schematic showing Chimera II (MC58/8047 A 174K) nucleotide and protein sequences. The sequence before the junction (cross-over) point is shown in lower case and the sequence following the junction point is shown in upper case. Lines of fifty residues are shown. Only the Neisserial sequences are shown; E. coli expression constructs contained an N-terminal Methionine and C-terminal hexa-histidine tag (LEHHHHHH). The A174K substitution is shown in bold and underlined text.
[0038] Figure 25 is a schematic showing Chimera lib (MC58/8047 A174K/K180R) nucleotide and protein sequences. The sequence before the junction (cross-over) point is shown in lower case and the sequence following the junction point shown in upper case. Lines of fifty residues are shown. Only the Neisserial sequences are shown; E. coli expression constructs contain an N-terminal Methionine and C-terminal hexa-histidine tag (LEHHHHHH). The A174K and Kl 8OR substitutions are shown in bold text. [0039] Figure 26 is a schematic showing Chimera III (NZ98254/8047) nucleotide and protein sequences. The sequence before the junction (cross-over) point is shown in lower case and the sequence following the junction point is shown in upper case. Lines of fifty residues are shown. Only the Neisserial sequences are shown; E. coli expression constructs contain an N-terminal Methionine and C-terminal hexa-histidine tag (LEHHHHHH). [0040] Figure 27 is a schematic showing Chimera IV (MC58/RM1090) nucleotide and protein sequences. The sequence before the junction (cross-over) point is shown in lower case and the sequence following the junction point is shown in upper case. Lines of fifty residues are shown. Only the Neisserial sequences are shown; E. coli expression constructs contain an N-terminal methionine and C-terminal hexa-histidine tag (LEHHHHHH). [0041] Figure 28 is a schematic showing Chimera V (NZ98254/RM1090) nucleotide and protein sequences. The sequence before the junction (cross-over) point is shown in lower case and the sequence following the junction point is shown in upper case. Lines of fifty residues are shown. Only the Neisserial sequences are shown; E. coli expression constructs contain an N-terminal methionine and C-terminal hexa-histidine tag (LEHHHHHH). [0042] Figure 29 provides a table showing positions of residues associated with JAR mAb binding. The reactive residue in fHBP was from the strain used as the source for immunization. For the anti-v.2 mAbs, the reactive strain is 8047, whose fHBP sequence is 99.6% identical to that from strain 2996. The non-reactive residue is that present in the non- reactive strain. Loss of reactivity associated with a change from the reactive to the non- reactive residue is indicated as knock-out (KO) and the converse change is indicated as knock-in (KI).
[0043] Figure 30 provides an image of a Western blot indicating residues involved in the JAR 10 and JAR 33 epitopes. E. coli lysates containing plasmids expressing the respective wild-type and mutant fHBPs were analyzed by Western blot with JAR 10 (Panel A), Penta-His mAb (Panel B), or JAR 33 (Panel C).
[0044] Figure 31 provides an image of a Western blot indicating a residue involved in the JAR 11, JAR 32 and JAR 35 epitopes. E. coli lysates containing plasmids expressing the respective wild-type and mutant fHBPs were analyzed by Western blot with JAR 32 (Panel A), JAR 35 (Panel B), JAR 11 (Panel C) or Penta-His mAb (Panel D). [0045] Figure 32 provides an image of a Western blot indicating residue involved in the JAR 13 epitope. E. coli lysates containing plasmids expressing wild-type and mutant fHBPs: lane 1, molecular weight marker; lane 2, pET21 (empty plasmid); lane 3, fHBP(8047)wt; lane 4, fHBP(8047)S216G; lane 5, fHBP(RM1090)wt; lane 6, fHBP(RM1090)G216S. Blots were probed with JAR 13 (Panel A) or Penta-His mAb (Panel B) and anti-mouse IgG-HRP secondary antibody.
[0046] Figure 33 provides an image of a Western blot of wildtype (WT) or Chimeric fHBP expressed in N. meningitidis. Lane 1, H44/76 KO fHBP transformed with pCom-fHBP v.2 WT plasmid; 2, H44/76 KO fHbp transformed with pCom-Chimera I plasmid; 3, Kaleidoscope marker; 4, Magic Mark marker; 5, H44/76 (v.l) WT cells; 6, 8047 (v.2) WT cells; 7, 8047 KO fHBP cells; 8, recombinant (r) fHBP v.l protein (gene from strain MC58); 9, rfHBP v.2 protein (gene from strain 8047). Upper panel, blot probed with anti-fHBP mAb JAR 3 (v.l); lower panel, blot probed with anti-fHBP mAb JAR 13 (v.2 or v.3). [0047] Figure 34 shows ribbon diagrams of full length v.1 fHBPs. Panel A, fHBP is partitioned into three domains indicated by various shades of gray. The A domain and the Ν- terminal portion of the B domain are on the left and the boundary between the A and B domains is indicated by an arrow at lysine 100. The C-terminal portion of the B domain together with the C domain is on the right, where the boundary between the two is designated by an arrow at glycine 164. Panel B, an alternative nomenclature describes the fHBP as having two structural domains. The Ν-terminal domain containing a mix of α helices and β strands is named the fHbpΝ domain (left) and the C-terminal domain consisting of β strands is labeled as the fHbpC domain (right). The fHbpΝ and the fHbpC are connected by a linker at or proximal to glycine 136. In some embodiments, the junction point relevant for the chimeric fHBP described herein is at or proximal to G136, indicated by an arrow in both panels. The models shown in both panels are constructed based on the ΝMR structure of Cantini et al. J Biol Chem 2009.
[0048] Before the present invention and specific exemplary embodiments of the invention are described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. [0049] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.
[0050] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
[0051] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antigen" includes a plurality of such antigens and reference to "the protein" includes reference to one or more proteins, and so forth. [0052] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS OF THE
INVENTION
[0053] The present disclosure provides chimeric fHBPs that can elicit antibodies that are bactericidal for different fHBP variant strains of N. meningitidis, and methods of use.
DEFINITIONS
[0054] "Factor H Binding Protein" (fHBP), which is also known in the literature as
GNA1870, GNA 1870, ORF2086, LP2086 (lipoprotein 2086), and "741" refers to a polypeptide of N. meningitidis that is a lipoprotein presented on the surface of the bacterium. N. meningitidis strains have been sub-divided into three fHBP variant groups (referred to as variant 1 (v.l), variant 2 (v.2), and variant 3 (v.3) in some reports (Masignani et al. 2003, supra) and Family A and B in other reports (see, e.g., Fletcher et al. 2004 Infect Immun 2088-2100)) based on amino acid sequence variability and immunologic cross-reactivity (Masignani et al. J Exp Med 2003; 197:789-99). For clarity, the present disclosure uses the v.l, v.2 and v.3 terminology. Because the length of variant 2 (v.2) fHBP protein (from strain 8047) and variant 3 (v.3) fHBP (from strain M1239) differ by -1 and +7 amino acid residues, respectively, from that of MC58, the numbering used to refer to residues for v.2 and v.3 fHBP proteins differs from numbering based on the actual amino acid sequences of these proteins. Thus, for example, reference to a leucine residue (L) at position 166 of the v.2 or v.3 fHBP sequence in Figure 3 refers to the residue at position 165 of the v.2 protein and at position 173 in the v.3 protein. For further clarification, see Figure 4 for alignment. [0055] The term "heterologous" refers to two components that are defined by structures derived from different sources. For example, where "heterologous" is used in the context of a chimeric polypeptide, the chimeric polypeptide includes operably linked amino acid sequences that can be derived from different polypeptides (e.g., a first component from a fHBP v.l polypeptide and a second component from a fHBP v.2 polypeptide). Similarly, "heterologous" in the context of a polynucleotide encoding a chimeric polypeptide includes operably linked nucleic acid sequence that can be derived from different genes (e.g., a first component from a nucleic acid encoding a fHBP v.l polypeptide and a second component from a nucleic acid encoding a fHBP v.2 polypeptide). Such chimeric polypeptides as described herein provide for presentation of epitopes in a single polypeptide that are normally found in different polypeptides. Other exemplary "heterologous" nucleic acids include expression constructs in which a nucleic acid comprising a coding sequence is operably linked to a regulatory element (e.g., a promoter) that is from a genetic origin different from that of the coding sequence (e.g., to provide for expression in a host cell of interest, which may be of different genetic origin relative to the promoter, the coding sequence or both). For example, a T7 promoter operably linked to a polynucleotide encoding a fHBP polypeptide or domain thereof is said to be a heterologous nucleic acid. "Heterologous" in the context of recombinant cells can refer to the presence of a nucleic acid (or gene product, such as a polypeptide) that is of a different genetic origin than the host cell in which it is present. For example, a Νeisserial amino acid or nucleic acid sequence of one strain is heterologous to a
Νeisserial host of another strain. [0056] "Heterologous" as used herein in the context of a chimeric fHBP (e.g.,
"heterologous fHBP domain", e.g., a "heterologous B domain", "heterologous C domain") indicates that the chimeric fHBP protein contains operably linked and contiguous amino acid sequences of structural elements of at least two different fHBP variants (e.g., so as to provide for presentation of epitopes of a v.1 fHBP, and presentation of a v.2 fHBP and/or a v.3 fHBP in a single fHBP polypeptide). For example, a "heterologous B domain" refers to a polypeptide which comprises a B domain that contains a first portion having a contiguous amino acid sequence of a B domain of a first fHBP variant operably linked to a second portion having a contiguous amino acid sequence of a B domain of a second fHBP variant. [0057] "Derived from" in the context of an amino acid sequence or polynucleotide sequence (e.g., an amino acid sequence "derived from" a v.l fHBP) is meant to indicate that the polypeptide or nucleic acid has a sequence that is based on that of a reference polypeptide or nucleic acid (e.g., a naturally occurring fHBP protein or encoding nucleic acid), and is not meant to be limiting as to the source or method in which the protein or nucleic acid is made. "Derived from" in the context of bacterial strains is meant to indicate that a strain was obtained through passage in vivo, or in in vitro culture, of a parental strain and/or is a recombinant cell obtained by modification of a parental strain.
[0058] "Conservative amino acid substitution" refers to a substitution of one amino acid residue for another sharing chemical and physical properties of the amino acid side chain (e.g., charge, size, hydrophobicity/hydrophilicity). "Conservative substitutions" are intended to include substitution within the following groups of amino acid residues: gly, ala; val, ile, leu; asp, glu; asn, gin; ser, thr; lys, arg; and phe, tyr. Conservative amino acid substitutions in the context of a chimeric fHBP disclosed herein are selected so as to preserve presentation of an epitope of interest. Guidance for such substitutions can be drawn from alignments of amino acid sequences of polypeptides presenting the epitope of interest. [0059] The term "protective immunity" means that a vaccine or immunization schedule that is administered to a mammal induces an immune response that prevents, retards the development of, or reduces the severity of a disease that is caused by Neisseria meningitidis, or diminishes or altogether eliminates the symptoms of the disease. Protective immunity can be accompanied by production of bactericidal antibodies. It should be noted that production of bactericidal antibodies against Neisseria meningitidis is accepted in the field as predictive of a vaccine's protective effect in humans. (Goldschneider et al., 1969, J. Exp. Med. 129:1307; Borrow et al. 2001 Infect Immun. 69:1568). [0060] The phrase "a disease caused by a strain of capsular group B of Neisseria meningitidis" encompasses any clinical symptom or combination of clinical symptoms that are present in an infection of a human with a member of capsular group B of Neisseria meningitidis. These symptoms include but are not limited to: colonization of the upper respiratory tract {e.g. mucosa of the nasopharynx and tonsils) by a pathogenic strain of capsular group B of Neisseria meningitidis, penetration of the bacteria into the mucosa and the submucosal vascular bed, septicemia, septic shock, inflammation, haemmorrhagic skin lesions, activation of fibrinolysis and of blood coagulation, organ dysfunction such as kidney, lung, and cardiac failure, adrenal hemorrhaging and muscular infarction, capillary leakage, edema, peripheral limb ischaemia, respiratory distress syndrome, pericarditis and meningitis. [0061] The phrase "broad spectrum protective immunity" means that a vaccine or immunization schedule elicits "protective immunity" against at least more than one strain (and can be against at least two, at least three, at least four, at least five, against at least eight, or more strains) of Neisseria meningitidis, wherein each of the strains expresses a different fHBP subvariant or fHBP variant. The present disclosure specifically contemplates and encompasses a vaccine or vaccination regimen that confers protection against a disease caused by a member of any capsular group (e.g., A, B, or C), with protection against disease caused by a capsular group B strain of Neisseria meningitidis being of interest due to the epidemiological prevalence of strains causing disease with this capsular group and lack of broadly effective group B vaccines.
[0062] The phrase "specifically binds to an antibody" or "specifically immunoreactive with", in the context of an antigen (e.g., a polypeptide antigen) refers to a binding reaction which is based on and/or is probative of the presence of the antigen in a sample which may also include a heterogeneous population of other molecules. Thus, under designated conditions, the specified antibody or antibodies bind(s) to a particular antigen or antigens in a sample and do not bind in a significant amount to other molecules present in the sample. "Specifically binds to an antibody" or "specifically immunoreactive with" in the context of an epitope of an antigen (e.g., an epitope of a polypeptide) refers to a binding reaction which is based on and/or is probative of the presence of the epitope in an antigen (e.g., polypeptide) which may also include a heterogeneous population of other epitopes, as well as a heterogeneous population of antigens. Thus, under designated conditions, the specified antibody or antibodies bind(s) to a particular epitope of an antigen and do not bind in a significant amount to other epitopes present in the antigen and/or in the sample. [0063] The phrase "in a sufficient amount to elicit an immune response" means that there is a detectable difference between an immune response indicator measured before and after administration of a particular antigen preparation. Immune response indicators include but are not limited to: antibody titer or specificity, as detected by an assay such as enzyme- linked immunoassay (ELISA), bactericidal assay, flow cytometry, immunoprecipitation, Ouchter-Lowny immunodiffusion; binding detection assays of, for example, spot, Western blot or antigen arrays; cytotoxicity assays, etc.
[0064] A "surface antigen" is an antigen that is present in a surface structure of
Neisseria meningitidis {e.g. the outer membrane, inner membrane, periplasmic space, capsule, pili, etc.).
[0065] "Isolated" refers to an entity of interest that is in an environment different from that in which the compound may naturally occur. "Isolated" is meant to include compounds that are within samples that are substantially enriched for the compound of interest and/or in which the compound of interest is partially or substantially purified." [0066] "Enriched" means that a sample is non-naturally manipulated (e.g., by an experimentalist or a clinician) so that a compound of interest is present in a greater concentration (e.g., at least a three-fold greater, at least 4-fold greater, at least 8-fold greater, at least 64-fold greater, or more) than the concentration of the compound in the starting sample, such as a biological sample (e.g., a sample in which the compound naturally occurs or in which it is present after administration), or in which the compound was made (e.g., as in a bacterial polypeptide, antibody, chimeric polypeptide, and the like) [0067] A "knock-out" or "knockout" of a target gene refers to an alteration in the sequence of the gene that results in a decrease of function of the target gene, e.g., such that target gene expression is undetectable or insignificant, and/or the gene product is not functional or not significantly functional. For example, a "knockout" of a gene involved in LPS synthesis indicates means that function of the gene has been substantially decreased so that the expression of the gene is not detectable or only present at insignificant levels and/or a biological activity of the gene product (e.g., an enzymatic activity) is significantly reduced relative to prior to the modification or is not detectable. "Knock-outs" encompass conditional knock-outs, where alteration of the target gene can occur upon, for example, exposure to a predefined set of conditions (e.g., temperature, osmolarity, exposure to substance that promotes target gene alteration, and the like. A "knock-in" or "knockin" of a target gene refers to a genetic alteration in a host cell genome that that results in an increase in a function provided by the target gene, FHBP AND FHBP-ENCODING NUCLEIC ACIDS
[0068] Before describing further exemplary chimeric fHBPs contemplated by the present disclosure, it is helpful to describe naturally-occurring fHBP from which the chimeric fHBPs may be derived.
For convenience and clarity, the native amino acid sequence of the v.l fHBP of the N. meningitidis strain MC58 was arbitrarily selected as a reference sequence for all native v.l, v.2, and v.3 fHBP amino acid sequences, as well as for the chimeric fHBPs described herein. Two nomenclature systems have been adopted to describe fHBP: one, which for convenience divided the protein into three domains, designated A, B and C (Giuliani et al., Infect Immun 2005; 73:1151-60), and the other based on three-dimensional structural data. In the alternative nomenclature system that describes fHBP based on three-dimensional structural data, fHBP is divided into two domains: the fHbpΝ and the fHbpC. Details of each of these domains with reference to the amino acid sequence of v.l fHBP of MC58 strain is described below.
A, B, and C domains using first definition.
[0069] As noted above, the nomenclature based on three domains describes fHBP as having an "A domain", a "B domain", and a "C domain". The amino acid sequence of the v.l fHBP of the MC58 strain along with the boundaries of the A, B and C domains is shown in Fig. 21. The QlOl and G164 residues indicated by the upward arrows denote the A/B and B/C domain boundaries, respectively. The "α" symbols indicate the position of the first and second α helices of the fHBP (referred to as AHl and AH2). Residues GEHT are underlined followed by the second α helix (AH2) of fHBP. Panel A in Fig. 34 also shows a ribbon diagram of the full length fHBP with the A, B, and C domains indicated as various shades of gray.
[0070] Fig. 3 provides a schematic of a truncated structural model of fHBP having operably linked B and C domains (the A domain and a portion of the Ν-terminal portion of the B domain are not shown). The native v.l fHBP of MC58 was again used as a reference sequence for purposes of residue numbering. Amino acid residues identified by site-directed mutagenesis of fHBP that contribute to binding of nine anti-fHBP mAbs (referred to as "JAR" mAbs) are noted. Coordinates from the solution structure of the B and C domains of fHBP from strain MC58 were used to construct the model. The α helix of the B domain is illustrated, as are the loops and β strands of the C domain.
Three-dimensional structural domains/fHbpN and fHbpC
[0071] In an alternative nomenclature system, fHBP is described as having two structural domains as opposed to the three domains described above. The two-domain nomenclature system is based on structural information of a full-length fHBP from which three-dimensional models may be constructed, such as the ones shown in Fig. 34. Structural modeling reveals that full-length fHBP is found to exist in solution as two separate domains connected by a linker. The amino acid sequence of the v.l fHBP of the MC58 strain is shown in Fig. 21 with end of the fHbpN domain indicated with an arrow at glycine 136. The N- terminal domain is named fHbpN (residues 8-136) and the C-terminal domain fHbpC (residues 141-255), each comprising at least 8 antiparallel β strands and joined by a native linker (residues 137-140). As seen in Fig. 21, the linker also comprises α-helix AH2 as "α" below the sequence in Fig. 21 marks the positions of α helices that reside in fHBP. For purposes of simplification herein, the fHbpC domain is considered to include the linker that connects the N-terminal and C-terminal domains based on the convention of this nomenclature.
[0072] fHBP has been divided into three variant groups (referred to as variant 1 (v.l), variant 2 (v.2), and variant 3 (v.3)) based on amino acid sequence variability and immunologic cross-reactivity (Masignani et al. 2003 J Exp Med 197:789-99). In certain studies, fHBP has also been subdivided into two sub-families designated sub-family A (which includes v.2 and v.3 of Masignani et al., 2003 J Exp Med 197:789-99) and sub-family B (v.l) (Fletcher et al., 2004, Infect Immun. 72: 2088-100). "Variant" as used in the context of an "fHBP variant" refers to an fHBP that share at least 89% amino acid sequence identity with the prototype strain of that variant group (strain MC58 for v.l; strain 2996 for v.2; and strain M1239 for v.3). These were the original prototype sequences described by Masignani et al., J. Exp. Med., 2003. Strains within a variant group encode fHBPs with greater than 88% amino acid identity, whereas strains of different fHBP variant groups range from approximately 60-88% identical. fHBPs in the same "variant" group possess greater than 88% identity to the respective prototype sequence (v.l, strain MC58; v.2, strain 2996; v.3, strain M1239). A "subvariant" as used in the context of an "fHBP subvariant" refers to fHBP polypeptides that differ from the prototype sequence. For example, strain NZ98/254 is referred to as an fHBP v.1 subvariant, with 91% identity to the prototype sequence from strain MC58; strain RM1090 is referred to as an fHBP v.2 subvariant, with a sequence that is 94% identical to the v.2 prototype strain 2996. Examples of subvariants, and their relative amino acid sequence identities, are provided in Figures 8 A and 8B. [0073] fHBP polypeptides, and encoding nucleic acids, from which portions of the chimeric fHBPs of the present disclosure can be derived may be from any suitable N. meningitidis strain. As is known in the art, N. meningitidis strains are divided into serologic groups (capsular groups), serotypes (PorB phenotypes) and subtypes (PorA phenotypes) on the basis of reactions with polyclonal (Frasch, C. E. and Chapman, 1973, /. Infect. Dis. 127: 149-154) or monoclonal antibodies that interact with different surface antigens. Capsular grouping traditionally has been based on immunologically detectable variations in the capsular polysaccharide but is being replaced by PCR of genes encoding specific enzymes responsible for the biosynthesis of the structurally different capsular polysaccharides. About 12 capsular groups (including A, B, C, X, Y, Z, 29-E, and W- 135) are known. Strains of the capsular groups A, B, C, Y and W- 135 account for nearly all meningococcal disease. Serotyping traditionally has been based on monoclonal antibody defined antigenic differences in an outer membrane protein called Porin B (PorB). Antibodies defining about 21 serotypes are currently known (Sacchi et al., 1998, Clin. Diag. Lab. Immunol. 5:348). Serosubtyping has been based on antibody defined antigenic variations on an outer membrane protein called Porin A (PorA). Both serotyping and serosubtyping are being replaced by PCR and/or DΝA sequencing for identification of genes encoding the variable regions of PorB and PorA, respectively that are associated with mAb reactivity (e.g. Sacchi, Lemos et al., supra; Urwin et al., 1998, Epidem. and Infect. 120:257).
[0074] N. meningitidis also may be divided into clonal groups or subgroups, using various techniques that directly or indirectly characterize the bacterial genome. These techniques include multilocus enzyme electrophoresis (MLEE), based on electrophoretic mobility variation of an enzyme, which reflects the underlying polymorphisms at a particular genetic locus. By characterizing the variants of a number of such proteins, genetic "distance" between two strains can be inferred from the proportion of mismatches. Similarly, clonality between two isolates can be inferred if the two have identical patterns of electrophoretic variants at number of loci. In more recent literature, multilocus sequence typing (MLST) has superseded MLEE as the method of choice for characterizing the microorganisms. Using MLST, the genetic distance between two isolates, or clonality, is inferred from the proportion of mismatches in the DΝA sequences of seven housekeeping genes in Neisseria meningitidis strains (Maiden et al., 1998, Proc. Natl. Acad. Sci. USA 95:3140). [0075] While N. meningitidis strains of any capsular group may be used, N. meningitidis strains of capsular group B are of particular interest as sources from which nucleic acid encoding fHBP and domains thereof are derived.
[0076] While the specification provides the amino acid sequence of exemplary fHBPs from which the chimeric fHBP can be derived, this is not intended to be limiting. For example, the chimeric fHBP can contain amino acid sequences that are at least 80%, at least 85%, at least 90%, or at least 95% identical to an amino acid sequence of a naturally- occurring fHBP.
[0077] Nucleic acids encoding fHBP polypeptides for use in construction of chimeric fHBPs contemplated herein are known in the art. Exemplary fHBP polypeptides are described in, for example, WO 2004/048404; Masignani et al. 2003 J Exp Med 197:789-799; Fletcher et al. Infect Immun 2004 2088-2100; Welsch et al. J Immunol 2004 172:5606-5615; and WO 99/57280. Nucleic acid (and amino acid sequences) for fHBP variants and subvariants are also provided in GenBank as accession nos.: NC_003112, GenelD: 904318 (NCBI Ref. NP_274866) (from N. meningitidis strain MC58); AY548371 (AAT01290.1) (from N. meningitidis strain CU385); AY548370 (AAT01289.1) (from N. meningitidis strain H44/76); AY548377 (AAS56920.1) (from N. meningitidis strain M4105); AY548376 (AAS56919.1) (from N. meningitidis strain M1390); AY548375 (AAS56918.1) (from N. meningitidis strain Ν98/254); AY548374 (AAS56917.1) (from N. meningitidis strain M6190); AY548373 (AAS56916.1) (from N. meningitidis strain 4243); and AY548372 (AAS56915.1) (from N. meningitidis strain BZ83).
[0078] For purposes of identifying relevant amino acid sequences contemplated for use in the chimeric fHBPs disclosed herein, it should be noted that the immature fHBP protein includes a leader sequence of about 19 residues. Furthermore, when provided an amino acid sequence the ordinarily skilled person can readily envision the sequences of nucleic that can encode for, and provide for expression of, a polypeptide having such an amino acid sequence.
[0079] In addition to the specific amino acid sequences and nucleic acid sequences provided herein, the disclosure also contemplates polypeptides and nucleic acids having sequences that are at least 80%, at least 85%, at least 90%, or at least 95% identical in sequence to such exemplary amino acid and nucleic acids. The terms "identical" or percent "identity," in the context of two or more polynucleotide sequences, or two or more amino acid sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (e.g., at least 80%, at least 85%, at least 90%, or at least 95% identical over a specified region), when compared and aligned for maximum correspondence over a designated region, e.g., a B domain or portion thereof, e.g., a region at least about 30, 35, 40, 45, 50, 55, 60, 65 or more amino acids or nucleotides in length, and can be up to the full-length of the reference amino acid or nucleotide sequence (e.g., a full-length fHBP). The disclosure specifically contemplates both naturally-occurring polymorphisms and synthetically produced amino acid sequences and their encoding nucleic acids.
[0080] For sequence comparison, typically one sequence acts as a reference sequence
(e.g., a naturally-occurring fHBP polypeptide sequence), to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer program, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
[0081] Examples of algorithms that are suitable for determining percent sequence identity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. MoI. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389- 3402, respectively. Software for performing BLAST analyses is publicly available through 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. [0082] In one embodiment, residue positions which are not identical differ by conservative amino acid substitutions. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine.. [0083] Sequence identity between two nucleic acids can also be described in terms of hybridization of two molecules to each other under stringent conditions. The hybridization conditions are selected following standard methods in the art (see, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N. Y.). An example of stringent hybridization conditions is hybridization at 5O0C or higher and 0.1 x SSC (15 mM sodium chloride/1.5 mM sodium citrate). Another example of stringent hybridization conditions is overnight incubation at 42 0C in a solution: 50 % formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1 x SSC at about 65 0C. Stringent hybridization conditions are hybridization conditions that are at least as stringent as the above representative conditions, where conditions are considered to be at least as stringent if they are at least about 80% as stringent, typically at least 90% as stringent as the above specific stringent conditions.
[0084] The chimeric fHBP of the present disclosure is described in more detail below in the context of both the nomenclature dividing the protein into three domains used by Giuliani et al. (Infect Immun 2005; 73:1151-60) and the three-dimensional structural nomenclature.
A Domain offHBPs
[0085] As noted above, fHBP may be described as having the following three domains to facilitate analysis: A domain, B domain, and C domain. As shown in Fig. 21, the upward arrows at QlOl and G164 demarcate the boundaries between A/B domains and B/C domains, respectively. The chimeric fHBPs of the present disclosure optionally include an A domain. For convenience and clarity, the A domain can be structurally defined as those residues corresponding to residues 1-100 of v.l fHBP of MC58, where the numbering is based on amino acid sequence of MC58 v.l fHBP lacking the signal sequence (Masignani et al., 2003 J Exp Med 197:789-99) (see Figure 21). As exemplified in the alignment of v.l fHBP of MC58 and v.2 fHBP of 8047, the respective amino acid sequences of the A domains of fHBPs normally share significant amino acid sequence identity (see Fig. 8B). Chimeric fHBPs which contain an A domain can contain a contiguous A domain amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to an amino acid sequence of an A domain of a naturally occurring fHBP. The A domain may be derived from the same variant group (and may be derived from the same fHBP) as the N-terminal portion of the B domain, such that the amino acid sequence at the A/B junction is one that may be found in nature. Alternatively, the A domain amino acid sequence may be derived from a fHBP variant group different from the fHBP variant group from which the N-terminal amino acid sequence of the B domain is derived (e.g., the A domain may be derived from a v.2 fHBP and the N-terminal amino acid sequence of the B domain derived from a v.l fHBP).
B Domain of v.l fHBP
[0086] As noted above, the chimeric fHBPs of the present disclosure contain amino acid sequence of a v.l fHBP B domain. Amino acid sequences of v.l fHBP, including v.l fHBP B domains, are well known in the art and can be used to derive the desired amino acid sequence of a chimeric fHBP disclosed herein. Fig. 13 provides the amino acid sequences of an N-terminal portion of the B domain of selected v.1 fHBPs. The alignment illustrates the position and identity of naturally occurring polymorphisms among v.l fHBPs. Figure 8 A illustrates the amino acid sequence identity between full length fHBPs of exemplary v.l, v.2 and v.3 strains, and further illustrates the presence or absence of epitopes defined by the indicated JAR mAbs. Figure 8B illustrates the amino acid sequence identity between the A, B, and C domains of exemplary v.l, v.2 and v.3 fHBPs, as well as amino acid sequence identity within the N-terminal (101-135) and C-terminal (136-164) portions of B domains. [0087] Fig. 21 shows the amino acid sequence of the fHBP of the v.l strain MC58, and illustrates the position and of a full-length B domain (defined by residues 101-164 and encompassing the amino acid sequence of GEHT followed by α -helix AH2). Fig. 22, Panel A illustrates that an N-terminal portion of the B domain of the v.1 fHBP of the MC58 strain, can encompass an amino acid sequence defined by residues corresponding to residues N- terminal of the GEHT residues and extending to the N-terminus of the B domain at a residue corresponding to residue 101. The C-terminal portion of the B domain of the v.1 fHBP of the MC58 strain can encompass an amino acid sequence defined by those residues corresponding to an amino acid sequence extending N-terminally from residue 164 of the B domain, and encompassing up to and including the amino acid sequence of GEHT. Thus, a full-length B domain is structurally defined by the residues corresponding to residues 101-164 of the v.l of fHBP of MC58, where residues 101-135 can define an exemplary N-terminal portion of the B domain and residues 136-164 can define an exemplary C-terminal portion of the B domain, where the numbering is based on amino acid sequence of MC58 v.l fHBP lacking the signal sequence (Masignani et al., 2003 J Exp Med 197:789-99).
[0088] As will be described below in more detail, it should be noted that in the context of chimeric fHBPs of the present disclosure having a heterologous B domain, the C- terminus of the N-terminal portion of the B domain (and thus the N-terminus of the C- terminal portion of the heterologous B domain) is defined by the position of the junction point, which can be present N-terminal or C-terminal to the amino acid sequence of GEHT, as discussed below in more detail. For example, the junction point of a heterologous B domain of a chimeric fHBP can be positioned C-terminal to a sequence corresponding to the GEHT, and thus can extend beyond a residue corresponding to residue 135. [0089] Exemplary chimeric fHBP include those comprising a B domain having a contiguous amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to an N-terminal B domain amino acid sequence of a v.l fHBP, e.g., at least 80%, at least 85%, at least 90%, or at least 95% identical to a contiguous amino acid sequence of the N-terminal B domain amino acid sequence exemplified in Fig. 13. Exemplary chimeric fHBP having a heterologous B domain contain at least 35, at least 40, at least 45, at least 50 residues (and in some embodiments no more than 50 residues) of a contiguous N-terminal amino acid sequence of a B domain of a v.l fHBP.
B and C domains of v.2 fHBP and of v.3 fHBP
[0090] Exemplary chimeric fHBPs of the present disclosure contain a heterologous B domain containing an N-terminal amino acid sequence derived from an N-terminal portion of a v.1 fHBP B domain and the remaining C-terminal portion derived from the corresponding C-terminal portion of a v.2 (or v.3) fHBP B domain, followed by the contiguous amino acid sequence of a v.2 (or v.3) C domain. For convenience and clarity, the C domain can be structurally defined as those residues corresponding to residues 165-255 of v.l fHBP of MC58, where the numbering is based on amino acid sequence of MC58 v.l fHBP lacking the signal sequence (Masignani et al., 2003 J Exp Med 197:789-99) (see Figure 21). [0091] Amino acid sequences of v.2 and v.3 fHBP, including v.2 and v.3 fHBP B and
C domains, are well known in the art and can be used to derive the desired amino acid sequence of a chimeric fHBP disclosed herein. Fig. 14 provides the amino acid sequences of a C-terminal portion of the B domain (as exemplified by the C-terminal 25 amino acids of v.2 fHBP B domain) and the full-length C domains of selected v.2 fHBPs. The alignment illustrates the position and identity of naturally occurring polymorphisms among v.2 fHBPs. [0092] Exemplary chimeric fHBP include those comprising a B domain containing a
C-terminal amino acid sequence derived from a v.2 or v.3 B domain, usually having a contiguous amino acid sequence that is greater than or at least 85%, at least 90%, or at least 95% identical to an C-terminal B domain amino acid sequence of a v.2 or v.3 fHBP, e.g., at least 80%, at least 85%, at least 90%, or at least 95% identical to a contiguous amino acid sequence of the N-terminal B domain amino acid sequence, such as those v.2 sequences exemplified in Fig. 14. Where the chimeric fHBP contains a heterologous C domain, the B domain can be at least 80%, at least 85%, at least 90%, or at least 95% identical to a contiguous amino acid sequence of a full-length B domain amino acid sequence of a v.1 fHBP. A full-length B domain of a v.l fHBP generally is about 64 residues in length. fHbpN Domain offHBPs
[0093] As discussed previously, fHBP may also be described has having two structural domains: fHbpN and fHbpC, based on an alternative nomenclature that is derived from the structure of the full-length fHBP. As shown in Fig. 21 and panel B of Fig. 32, glycine 136 marks approximately the beginning of a linker between the N-terminal and the C- terminal domains, named the fHbpN and fHbpC domains, respectively. The chimeric fHBP of the present disclosure may include a full-length fHbpN domain or a partial fHbpN domain. For convenience and clarity, the fHbpN domain can be structurally defined as those residues corresponding to residues 1-136 of v.l fHBP of MC58, where the numbering is based on amino acid sequence of MC58 v.l fHBP lacking the signal sequence (Masignani et al., 2003 J Exp Med 197:789-99) (see Fig. 21). As exemplified in the alignment of v.l fHBP of MC58 and v.2 fHBP of 8047, the respective amino acid sequences of the first 100 residues of the fHbpN domain normally share significant amino acid sequence identity (Fig. 8B). Chimeric fHBP which contains an fHbpN domain can contain a contiguous fHbpN domain amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to an amino acid sequence of an fHbpN domain of a naturally occurring fHBP.
[0094] Alternatively, the chimeric fHBP may include a partial fHbpN domain, such that an N-terminal portion of the fHbpN is truncated. The partial fHbpN domain may comprise at least 30, 40, or 50 of a contiguous C-terminal amino acid sequence of the full length fHbpN domain.
[0095] Exemplary chimeric fHBPs include those comprising a full or partial fHbpN domain having a contiguous amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to at least a C-terminal portion of the fHbpN amino acid sequence of a v.l fHBP, e.g., at least 80%, at least 85%, at least 90%, or at least 95% identical to a contiguous amino acid sequence of the C-terminal fHbpN amino acid sequence exemplified in Fig. 13. An exemplary chimeric fHBP having heterologous domains contains at least 35, at least 40, at least 45, at least 50 residues (and in some embodiments no more than 50 residues) of a contiguous C-terminal amino acid sequence of an fHbpN domain of a v.l fHBP. [0096] The full or partial fHbpN domain may be derived from the same variant group
(and may be derived from the same fHBP) as certain portions of the fHbpC domain. Alternatively, the fHbpN amino acid sequence may be derived from a fHBP variant group different from the fHBP variant group from which the N-terminal amino acid sequence of the fHbpC domain is derived (e.g., the fHbpC domain may be derived from a v.2 fHBP and the C-terminal amino acid sequence of the fHbpN domain derived from a v.l fHBP). [0097] As noted above, the chimeric fHBPs of the present disclosure contain amino acid sequence of a v.l fHbpN domain. Amino acid sequences of v.l fHBP, including v.l fHbpN domains, are well known in the art and can be used to derive the desired amino acid sequence of a chimeric fHBP disclosed herein. Fig. 13 provides the C-terminal amino acid sequences of the fHbpN domain of selected v.l fHBPs. The alignment illustrates the position and identity of naturally occurring polymorphisms among v.l fHBPs. Figure 8A illustrates the amino acid sequence identity between full length fHBPs of exemplary v.l, v.2 and v.3 strains, and further illustrates the presence or absence of epitopes defined by the indicated JAR mAbs. Figure 8B illustrates the amino acid sequence identity between exemplary v.l, v.2 and v.3 fHBPs, as well as amino acid sequence identity within the C-terminal portion of the fHbpN (101-135) and N-terminal (136-164) portion of the fHbpC domains.
The junction between heterologous domains of a chimeric fHBP
[0098] As will be described below in more detail, it should be noted that in the context of chimeric fHBPs of the present disclosure having heterologous domains, the position of the junction point between heterologous domains can be present within or proximal to the linker that connects the fHbpN and the fHbpC domains. Glycine 136 defines the boundary between fHbpN and fHbpC and also marks the beginning of the linker sequence. The linker sequence corresponds approximately to residues 136 to 149 and includes α-helix AH2. For example, the junction point between heterologous domains of a chimeric fHBP can be positioned C-terminal to a sequence corresponding to the GEHT sequence underlined in Fig. 21. In some embodiments, the junction may be no more than 20, no more than 15, no more than 5 or less amino acid residues away from the amino acid sequence of GEHT or the linker sequence. In other embodiments where heterologous domains are present in the fHbp C domain, the junction between the heterologous domains may be positioned C-terminal to glycine 164. Glycine 164 is also indicated by an arrow in Fig. 21. fHbpC domain of v.2 fHBP and of v JfHBP
[0099] Exemplary chimeric fHBPs of the present disclosure contain heterologous domains comprising a full or partial fHbpN domain of a v.l fHBP fHbpN domain and a fHbpC domain derived from the fHbpC of a v.2 (or v.3) fHBP. For convenience and clarity, the fHbpC domain can be structurally defined as those residues corresponding to residues 141-255 of v.l fHBP of MC58, where the numbering is based on amino acid sequence of MC58 v.l fHBP lacking the signal sequence (Masignani et al., 2003 J Exp Med 197:789-99) (Fig. 21).
[00100] Amino acid sequences of v.2 and v.3 fHBP, including v.2 and v.3 fHBP fHbpN and fHbpC domains, are well known in the art and can be used to derive the desired amino acid sequence of a chimeric fHBP disclosed herein. Fig. 14 provides the amino acid sequences of the full-length fHbpC domains of selected v.2 fHBPs. The alignment illustrates the position and identity of naturally occurring polymorphisms among v.2 fHBPs. Exemplary chimeric fHBPs include those comprising an fHbpC amino acid sequence derived from a v.2 or v.3 fHbpC domain, usually having a contiguous amino acid sequence that is greater than or at least 85%, at least 90%, or at least 95% identical to the amino acid sequence of the fHbpC domain of a v.2 or v.3 fHBP, e.g., at least 80%, at least 85%, at least 90%, or at least 95% identical to a contiguous amino acid sequence of the fHbpC domain amino acid sequence, such as those v.2 sequences exemplified in Fig. 14.
[00101] In certain cases, instead of having the amino acid sequence of fHbpN derived from one variant and that of fHbpC derived from a different variant, fHbpC domain may contain two contiguous amino acid sequences derived from different variants. In cases where fHbpC contains heterologous sequences, a contiguous N-terminal amino acid sequence of fHbpC can be at least 80%, at least 85%, at least 90%, or at least 95% identical to a contiguous amino acid sequence of the corresponding amino acid sequence of a v.l fHBP.
CHIMERIC FACTOR H BINDING PROTEINS
[00102] As explained previously, fHBP may be described in the context of the three domains assigned by Giuliani et al (Infect Immun 2005 ; 73 : 1151 -60) or in the context of two three-dimensional structural domains. For the sake of brevity, the disclosure will adopt the nomenclature of the three domains, designated A, B, and C domains. However, all discussion in the context of the three functional domains can be readily understood in the context of the two structural domains based on what has been detailed above. [00103] As set out above, the chimeric fHBPs of the present disclosure generally include either a heterologous B domain and a C domain; or a B domain and a heterologous C domain. Such chimeric fHBPs are constructed so as to contain epitopes that elicit bactericidal antibodies effective against N. meningitidis strains producing more than one fHBP variant. [00104] The term "chimeric factor H binding protein" or "chimeric fHBP" refers to a polypeptide comprising, from Ν-terminus to C-terminus, an amino acid sequence of a B domain and of a C domain, wherein at least one of the B domain and the C domain contains a heterologous amino acid sequence characterized as having an Ν-terminal portion derived from a contiguous amino acid sequence of a v.l fHBP with the remaining B and C-terminal portion (or C terminal portion) being derived from a contiguous amino acid sequence of a v.2 or v.3 fHBP. The B domain and/or C domain amino acid sequences are generally derived from a contiguous amino acid sequence of a naturally-occurring fHBP and mutants thereof that maintain or introduce desired epitopes. Chimeric fHBP can optionally include an amino acid sequence of an fHBP A domain operably linked and Ν-terminal to the B domain. Chimeric fHBP can further optionally include a leader sequence, e.g., to provide for expression of the chimeric fHBP on a cell surface of a bacterial host cell. [00105] Where the chimeric fHBP contains a heterologous B domain, the heterologous
B domain generally comprises at least an Ν-terminal portion derived from a contiguous amino acid sequence of a v.l fHBP B domain and a C-terminal portion derived from a contiguous amino acid sequence of a v.2 or v.3 B domain, with the heterologous B domain being operably linked to a C domain derived from a contiguous amino acid sequence of a v.2 or v.3 fHBP C domain. Thus, for example, such chimeric fHBP can be described as having a heterologous B domain composed of an Ν-terminal portion for which a corresponding contiguous amino acid sequence of a v.l fHBP B domain sequence serves as a scaffold, and a C-terminal portion for which a corresponding contiguous amino acid sequence of a v.2 or v.3 fHBP B domain sequence serves as a scaffold.
[00106] As noted above, exemplary chimeric fHBP having a heterologous B domain contain at least 35, at least 40, at least 45, at least 50 residues (and in some embodiments no more than 50 residues) of a contiguous Ν-terminal amino acid sequence of a B domain of a v.l fHBP.
[00107] Where the chimeric fHBP contains a heterologous C domain, the B domain of the chimeric fHBP comprises a contiguous amino acid sequence of a v.l fHBP B domain operably linked to heterologous C domain comprising at least an Ν-terminal portion of a v.1 fHBP C domain and a C-terminal portion of a v.2 or v.3 C domain. Exemplary chimeric fHBP of this embodiment contain 2, 4, 6, 8 residues of an N-terminal sequence of a v.l C domain, with the remainder of the C domain being derived from a v.2 or v.3 C domain amino acid sequence.
[00108] Chimeric fHBP contemplated by the present disclosure include those having an amino acid sequence corresponding to a full-length B domain and a full-length C domain, and, optionally, a full-length A domain wherein the chimeric fHBP includes at least a heterologous B domain or a heterologous C domain. Other embodiments include chimeric fHBP having an amino acid sequence corresponding to a fragment of an A domain composed of a contiguous amino acid sequence encompassing amino acid defining an epitope bound by the JAR 4 mAb. Further embodiments include chimeric fHBP in which the C domain is truncated at the C-terminus, with the proviso that epitopes of interest (e.g., one or more of the epitopes bound by mAbs JAR 10, JAR 11, JAR 33, JAR 32/35, and JAR 13) are preserved so as to retain the ability to elicit antibodies that bind these epitopes. Chimeric fHBP also include those that lack an A domain, and have an N-terminally truncated B domain, with the proviso that the truncated B domain maintains expression of an epitope(s) of interest. Chimeric fHBP include those having a B domain that expresses an epitope bound by the JAR 5 mAb.
[00109] Chimeric polypeptides described herein can include additional heterologous amino acid sequences, e.g., to provide an N-terminal methionine or derivative thereof (e.g., pyroglutamate) as a result of expression in a bacterial host cell (e.g., E. coli) and/or to provide a chimeric polypeptide having a fusion partner at its N-terminus or C-terminus. Fusion partners of interest include, for example, glutathione-S-transferase (GST), maltose binding protein (MBP), His6-tag, and the like, as well as leader peptides from other proteins, particularly lipoproteins. Fusion partners can provide for additional features, such as in facilitating isolation and purification of the chimeric polypeptide.
[00110] Native fHBP usually contains an N-terminal cysteine to which a lipid moiety can be covalently attached. This cysteine residue is usually lipidated in the naturally- occurring protein, and can be lipidated in the chimeric fHBPs disclosed herein. Thus, in the amino acid sequences described herein (including those presented in any Sequence Listing), reference to "cysteine" or "C" at this position specifically includes reference to both an unmodified cysteine as well as to a cysteine that is lipidated (e.g., due to post-translational modification). Thus, the chimeric fHBP can be lipidated or non-lipidated. Methods for production of lipidated proteins in vitro, (see, e.g., Andersson et al., 2001 J. Immunological
Methods 255(1-2): 135-48) or in vivo are known in the art. For example, lipidated fHBP previously has been purified from the membrane fraction of E. coli protein by detergent extraction (Fletcher et al., 2004 Infection and Immunity 72(4):2088-100), which method may be adapted for the production of lipidated chimeric fHBP. Lipidated proteins may be of interest as such can be more immunogenic than soluble protein (see, e.g., Fletcher et al., 2004 Infection and Immunity 72(4):2088-100). [00111] Exemplary chimeric fHBPs are described in detail below.
Exemplary chimeric fHBPs
[00112] The chimeric fHBPs of the present disclosure encompass those that can be described in terms of one or more of, for example, the site at which heterologous sequences are joined within the chimeric fHBP (i.e., the "junction point"), the presence of epitopes specifically bound by a mAb, amino acid sequence, or any combination of such features that may be present in exemplary fHBPs.
Junction point of chimeric fHBP
[00113] In general, the junction point of the chimeric fHBP is the point at which amino acid sequence of the chimeric fHBP shifts from being derived from a contiguous amino acid sequence of a v.l fHBP to being derived from contiguous amino acid sequence of a v.2 or v.3 fHBP. The junction point thus provides for an amino acid sequence that is heterologous, i.e., derived from different fHBPs. The N-terminal portion and the C-terminal portions of a heterologous domain (i.e., heterologous B domain or heterologous C domain) of chimeric fHBP are joined at a junction point, with the junction point thus defining the length of the N- terminal and C-terminal portions of the chimeric domain that are derived from a v.1 or v.2/v.3 amino acid sequence.
[00114] In general, a B domain amino acid sequence comprising an amino acid sequence N-terminal to the second α helix of fHBP, which includes residues corresponding to those implicated in defining the JAR 5 mAb epitope (i.e., residues at positions 121 and 122 of a B domain v.l fHBP MC58, which are glycine and lysine, respectively) is denoted as the "N-terminal portion of the B domain" (see, e.g., Fig. 13, Fig. 21 and Fig. 22, Panel A). The amino acid sequence flanking and C-terminal to the N-terminal portion of the B domain is the "C-terminal (or distal) portion of the B domain" and is derived from a contiguous amino acid sequence of a v.2 or v.3 fHBP (Fig. 14 and Fig. 22). Together, the N-terminal and C-terminal portions of the B domain compose a heterologous B domain of a chimeric fHBP of the present disclosure. [00115] Where the chimeric fHBP has a heterologous B domain, the junction point may be positioned at a residue adjacent to the second α helix (AH2) (e.g., adjacent and C- terminal to a residue corresponding to residue 121 or 122 of Figure 21, e.g., adjacent and C- terminal to one of the residues of GEHTSFDK, e.g., adjacent and C-terminal to one of the residues of GEHT, N-terminal to AH2), or at a position C-terminal to AH2. [00116] In one embodiment, the junction point of the heterologous B domain can be positioned at any site corresponding to a site after the glycine residue or after the lysine residue, that define a JAR 5 monoclonal antibody (mAb) epitope of a v.1 fHBP (which residue is positioned within the B domain, i.e., at G121 or K122 of v.l fHBP strain MC58 reference sequence) but before a residue corresponding to a residue defining a JAR 11 mAb epitope of a v.2 fHBP (which residue is positioned in the C domain, i.e., A174 of v.2 fHBP strain 8047 reference sequence). In a related embodiment, the heterologous B domain is provided such that the JAR 5 mAb epitope, the JAR 11 epitope, or both the JAR 5 and JAR 11 epitopes are maintained such that the chimeric fHBP is specifically bound by the respective mAb.
[00117] In one embodiment, the junction point is positioned so that the chimeric fHBP contains a heterologous B domain, which has an N-terminal portion composed of a contiguous amino acid sequence of an N-terminal portion of a B domain of a v.1 fHBP containing a JAR 5 epitope (defined in part by G121 of v.l fHBP strain MC58) with the remaining portion (i.e., the C-terminal portion) of the B domain derived from a contiguous amino acid sequence of the corresponding C-terminal portion of a v.2 or v.3 fHBP B domain. The heterologous B domain is operably linked to a C domain derived from a contiguous amino acid sequence of a v.2 or v.3 fHBP, which can be the same or different v.2 or v.3 fHBP as that from which the C-terminal portion of the B domain is derived. [00118] Exemplary heterologous B domains include those at least 80% identical, at least 85% identical, at least 90% identical, at least 99% identical or more to a contiguous amino acid sequence of a v.l fHBP corresponding to residues 101-121, 101-122, 101-123, 101-124, 101-125, 101-126, 101-127, 101-128, 101-129, 101-130, 101-131, 101-132, 101- 133, 101-134, 101-134, 101-136, 101-137, 101-138, or 101-139 of a v.l fHBP amino acid sequence, where the numbering is based on MC58 fHBP as a reference. Such heterologous B domains include those having an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 99% identical or more to a contiguous amino acid sequence of a v.2 or v.3 fHBP so as to provide the remainder of the heterologous B domain having a C-terminus corresponding to residue 164 (again, using MC58 fHBP as a reference sequence of purposes of numbering).
[00119] For example, where the heterologous B domain includes residues 101-122 or a v.l fHBP, the C-terminal portion of the heterologous B domain includes residues 123-164 of a v.2 or v.3 fHBP. Accordingly, the C-terminal portion of the heterologous B domain can include an amino acid sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 99% identical or more to a contiguous amino acid sequence of a v.2 or v.3 fHBP corresponding to residues 122-164, 123-164, 124-164, 125-164, 126-164,127-164, 128-164, 129-164, 130-164, 131—164, 132-164, 133-164, 134-164, 135-164, 136-164, 137- 164, 139-164, or 140-164, where the N-terminal portion of the heterologous B domain is provided by the v.l sequences exemplified above.
[00120] In another embodiment, the junction point is positioned N-terminal to the second α helix (AH2), which are denoted in Figure 22 by "α". As pointed out above, the residues GEHT are highly conserved across v.l, v.2, and v.3 fHBP variants, and thus can serve as convenient junction point residues, as well as a convenient reference for the position of a junction point in a chimeric fHBP. For example, the junction point can be positioned within 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 residue(s) N-terminal to GEHT to provide a heterologous B domain (e.g., positioned at a site not more than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 residue N-terminal of GEHT); or is positioned within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 residues C-terminal to GEHT (e.g., positioned at a site not more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 residues C-terminal of GEHT), where a junction point at a site less than or equal to 26 residues C-terminal to GEHT provides a heterologous B domain and a junction point positioned at more than 26 residues C-terminal to GEHT produces a chimeric fHBP having a heterologous C domain.
[00121] For example, the junction point of the heterologous B domain can be selected such that the heterologous B domain amino acid sequence positioned N-terminal and flanking the amino acid sequence GEHT is derived from a v.l fHBP B domain amino acid sequence and the heterologous B domain amino acid sequence positioned C-terminal and flanking the GEHT is derived from a v.2 or v.3 fHBP B amino acid sequence. [00122] In some embodiments, the junction point is positioned so as to provide a heterologous B domain comprising an amino acid sequence that is greater than 80% (e.g., at least 81%), at least 85%, at least 90%, at least 95% or identical to an amino acid sequence of
[00123] QSHSALTAFQ TEQIQDSEHS GK (SEQ ID NO: 1) where the amino acid sequence optionally provides for an epitope that mediates specific binding of a JAR 5 mAb. Exemplary amino acid substitutions of the above sequence are as follows: QSHSALTA(F/L)Q TEQ(I/V/E)QD(S/P)E(H/D)S (GZEZR)K.
[00124] Exemplary modifications of the amino acid sequences of the heterologous B domain as set out above include, for example, one or more of the following substitutions of
SEQ ID NO:1 as follows: leucine (L) for the phenylalanine (F) at a residue corresponding to position 9; valine (V) or glutamic acid (E) for isoleucine (I) at residue position 14; proline (P) for serine (S) at residue position 17; aspartic acid (D) for histidine (H) at residue position 19; arginine (R) for glutamine (Q) at residue position 28; valine (V) for alanine (A) at residue position 35; glycine (G) for aspartic acid (D) at residue position 42; or lysine (K) for glutamic acid (E) at residue position 46.
[00125] In further embodiments, the heterologous B domain comprises an amino acid sequence represented by the formula:
QSHSALTA(F/L)Q TEQ(I/V/E)QD(S/P)E(H/D)S (G/E/R)KMVAKR(Q/R)FR
IGDI(A/V)GEHTA FNQLP (D/S)
[00126] In some embodiments, the junction point is positioned so as to provide a heterologous B domain comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% or identical to an amino acid sequence of
TEQIQDSEHS GKMVAKRQFR IGDIAGEHTA FNQLPD, where the amino acid sequence optionally provides for an epitope that mediates specific binding of a JAR 5 mAb.
[00127] In other embodiments the heterologous B domain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% or identical to an amino acid sequence of
QSHSALTAFQ TEQIQDSEHS GKMVAKRQFR IGDIAGEHTA FNQLPD where the amino acid sequence optionally provides for an epitope specifically bound by JAR
5 MAB. [00128] In still other embodiments, the heterologous B domain comprises a sequence at least 80%, at least 85%, at least 90%, at least 95% or identical to an amino acid sequence set out in Fig. 13, which provides an alignment of exemplary chimeric fHBPs III, IV and V sequences in the region of the junction point, which is indicated by the box. The residue, G121, implicated in the JAR 5 epitope is shown in bold and underlined. [00129] In some embodiments, the junction point is positioned so as to provide a heterologous B domain comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% or identical to an amino acid sequence of:
LTAFQ TEQIQDSEHS GKMVAKRQFR IGDIA where the amino acid sequence optionally provides for an epitope that mediates specific binding of a JAR 5 mAb.
[00130] In another embodiment, the junction point of the chimeric fHBP is positioned so that the chimeric fHBP contains a heterologous C domain composed of a contiguous amino acid sequence of an N-terminal portion of a C domain of a v.1 fHBP and a contiguous amino acid sequence of a C-terminal portion of a C domain of a v.2 or v.3 fHBP. [00131] For example, the junction point of a chimeric fHBP having a heterologous C domain can be in the loop regions of the β-barrel of the C domain, or in any highly conserved segment, for example at residues D160 or 1170. In one embodiment, the heterologous C domain includes an N-terminal sequence of KLTYTIDFA.
[00132] Exemplary chimeric fHBP are provided in the Examples below. The present disclosure contemplates these exemplary chimeric fHBP, as well as chimeric fHBP having at least 85%, at least 90%, at least 95% or greater amino acid sequence identity to the amino acid sequences of these exemplary chimeric fHBP (e.g., Chimera I, Chimera II, Chimera lib, Chimera III, Chimera IV, and Chimera V). The amino acid and nucleic acid sequences encoding Chimera I, Chimera II, Chimera lib, Chimera III, Chimera IV, and Chimera V are provided in Figures 23, 24, 25, 26, 27, and 28, respectively. The asterisk denotes the C- terminus of the amino acid sequence (corresponding to the stop codon of the encoding nucleic acid).
Inclusion or maintenance of epitope pairs that elicit antibodies that exhibit enhanced bactericidal activity when both are bound
[00133] Chimeric fHBP encompass chimeric fHBP that contain pairs of epitopes that elicit antibodies that, when both are bound to their respective epitopes, exhibit enhanced bactericidal activity against N. meningitidis than when either one is bound alone. Chimeric fHBP can be designed so as to ensure that such epitopes are maintained or to introduce such epitopes (e.g., by modification of an fHBP amino acid sequence to include a pair of epitopes heterologous to that fHBP amino acid sequence).
[00134] In general (and subject to the exception below), the distance between epitopes of such epitope pairs is selected so as to be less than 27 A but more than 14 A, and are usually located within a distance of about 18 A - 20 A. As discussed in the Examples below, a greater bactericidal effect was observed when two antibodies bound epitopes located at distances within these parameters. Without being held to theory, when bound by their respective antibodies, the distance between the epitopes is sufficient to facilitate interaction of the antibodies with factors of the complement cascade, but not so close as to result in inhibition of binding due to steric hindrance. Chimeric fHBP containing such epitopes for v.2 and v.3 strains can thus provide for production of antibodies having greater bactericidal activity against such strains. Examples of such epitope pairs are those epitopes bound by JAR 10 and JAR 11 (fHBP v.2); and by JAR 33 and JAR 32/ 35 (JAR 32 and JAR 35 bind to the same or overlapping epitopes) (fHBP v.3).
[00135] Chimeric fHBPs can also include epitope pairs where one epitope of the pair is defined by binding by the mAb JAR 4 and the second epitope is bound by an antibody that inhibits fH binding. Such an epitope pair is not necessarily subject to the constraints on distance between the epitopes as discussed above, with the proviso that the epitopes are not so close as to inhibit binding of their respective antibodies. As discussed in the Examples below, binding of an antibody that inhibits fH binding can be bactericidal along with another mAb that does not inhibit fH binding. For example, a mAb pair such as JAR 4 along with any anti-v.l or v.2 mAb that blocks fH binding can provide for production of antibodies that have enhanced bactericidal activity.
Inclusion or maintenance ofevitoves that elicit antibodies that inhibit fH binding
[00136] Chimeric fHBPs can be designed so as to include an epitope(s) that elicits antibodies that, when bound to fHBP, inhibit fH binding. For example, as set out below, when the epitopes bound by JAR 13 (v.2 epitope), JAR 11 (v.2 epitope), and JAR 32/35 (v.3 epitope) are bound by antibody, binding of fHBP to fH is inhibited. Thus, the presence of such fH-binding epitopes in the chimeric fHBP polypeptides can provide for production of antibodies that can facilitate protection through this pathway. Nucleic acid encoding chimeric fHBP
[00137] The chimeric fHBP can be generated using recombinant techniques to manipulate nucleic acids of different fHBPs known in the art to provide constructs encoding a chimeric fHBP of interest. As noted above, nucleic acids encoding a variety of different v.l, v.2, and v.3 fHBPs of N. meningitidis are available in the art, and their nucleotide sequences are known.
[00138] Amino acid and nucleic acid sequences of exemplary chimeric fHBPs are provided in Figures 23-28. It will be appreciated that the nucleotide sequences encoding the chimeric fHBPs can be modified so as to optimize the codon usage to facilitate expression in a host cell of interest (e.g., E. coli, N. meningitidis, human (as in the case of a DΝA-based vaccine), and the like). Methods for production of codon optimized sequences are known in the art.
METHODS OF PRODUCTION
[00139] Chimeric fHBPs can be produced by any suitable method, including recombinant and non-recombinant methods (e.g., chemical synthesis). Where the chimeric fHBP is produced using recombinant techniques, the methods can involve any suitable construct and any suitable host cell, which can be a prokaryotic or eukaryotic cell, usually a bacterial or yeast host cell, more usually a bacterial cell. Methods for introduction of genetic material into host cells include, for example, transformation, electroporation, conjugation, calcium phosphate methods and the like. The method for transfer can be selected so as to provide for stable expression of the introduced chimeric fHBP -encoding nucleic acid. The chimeric fHBP -encoding nucleic acid can be provided as an inheritable episomal element (e.g., plasmid) or can be genomically integrated.
[00140] Suitable vectors for transferring chimeric fHBP-encoding nucleic acid can vary in composition. Integrative vectors can be conditionally replicative or suicide plasmids, bacteriophages, and the like. The constructs can include various elements, including for example, promoters, selectable genetic markers (e.g., genes conferring resistance to antibiotics (for instance kanamycin, erythromycin, chloramphenicol, or gentamycin)), origin of replication (to promote replication in a host cell, e.g., a bacterial host cell), and the like. The choice of vector will depend upon a variety of factors such as the type of cell in which propagation is desired and the purpose of propagation. Certain vectors are useful for amplifying and making large amounts of the desired DNA sequence. Other vectors are suitable for expression in cells in culture. Still other vectors are suitable for transfer and expression in cells in a whole animal. The choice of appropriate vector is well within the skill of the art. Many such vectors are available commercially.
[00141] In one embodiment, the vector is an expression vector based on episomal plasmids containing selectable drug resistance markers and elements that provide for autonomous replication in different host cells (e.g., in both E. coli and N. meningitidis). One example of such a "shuttle vector" is the plasmid pFPIO (Pagotto et al. Gene 2000 244:13- 19).
[00142] Constructs can be prepared by, for example, inserting a polynucleotide of interest into a construct backbone, typically by means of DΝA ligase attachment to a cleaved restriction enzyme site in the vector. Alternatively, the desired nucleotide sequence can be inserted by homologous recombination or site-specific recombination. Typically homologous recombination is accomplished by attaching regions of homology to the vector on the flanks of the desired nucleotide sequence, while site-specific recombination can be accomplished through use of sequences that facilitate site-specific recombination (e.g., cre-lox, att sites, etc.). Nucleic acid containing such sequences can be added by, for example, ligation of oligonucleotides, or by polymerase chain reaction using primers comprising both the region of homology and a portion of the desired nucleotide sequence.
[00143] Vectors can provide for extrachromosomal maintenance in a host cell or can provide for integration into the host cell genome. Vectors are amply described in numerous publications well known to those in the art, including, e.g., Short Protocols in Molecular Biology, (1999) F. Ausubel, et al., eds., Wiley & Sons. Vectors may provide for expression of the nucleic acids encoding a chimeric fHBP, may provide for propagating the subject nucleic acids, or both.
[00144] Exemplary vectors that may be used include but are not limited to those derived from recombinant bacteriophage DNA, plasmid DNA or cosmid DNA. For example, plasmid vectors such as pBR322, pUC 19/18, pUC 118, 119 and the M13 mp series of vectors may be used. pET21 is also an expression vector that may be used. Bacteriophage vectors may include λgtlO, λgtl l, λgtl8-23, λZAP/R and the EMBL series of bacteriophage vectors. Further vectors that may be utilized include, but are not limited to, pJB8, pCV 103, pCV 107, pCV 108, pTM, pMCS, pNNL, pHSG274, COS202, COS203, pWE15, pWE16 and the charomid 9 series of vectors. [00145] For expression of a chimeric fHBP of interest, an expression cassette may be employed. Thus, the present disclosure provides a recombinant expression vector comprising a subject nucleic acid. The expression vector provides transcriptional and translational regulatory sequences, and may provide for inducible or constitutive expression, where the coding region is operably linked under the transcriptional control of the transcriptional initiation region, and a transcriptional and translational termination region. These control regions may be native to an fHBP from which the chimeric fHBP is derived, or may be derived from exogenous sources. In general, the transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. Promoters can be either constitutive or inducible, and can be a strong constitutive promoter (e.g., T7, and the like).
[00146] Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding proteins of interest. A selectable marker operative in the expression host may be present to facilitate selection of cells containing the vector. In addition, the expression construct may include additional elements. For example, the expression vector may have one or two replication systems, thus allowing it to be maintained in organisms, for example in mammalian or insect cells for expression and in a procaryotic host for cloning and amplification. In addition the expression construct may contain a selectable marker gene to allow the selection of transformed host cells. Selection genes are well known in the art and will vary with the host cell used.
[00147] It should be noted that chimeric fHBP of the present disclosure may comprise additional elements, such as a detectable label, e.g., a radioactive label, a fluorescent label, a biotin label, an immunologically detectable label (e.g., an HA tag, a poly-Histidine tag) and the like. Additional elements of chimeric fHBP can be provided to facilitate isolation (e.g., biotin tag, immunologically detectable tag) through various methods (e.g., affinity capture, etc.). Chimeric fHBP can optionally be immobilized on a support through covalent or non- covalent attachment.
[00148] Isolation and purification of chimeric fHBP can be accomplished according to methods known in the art. For example, chimeric fHBP can be isolated from a lysate of cells genetically modified to express a chimeric fHBP, or from a synthetic reaction mix, by immunoaffinity purification, which generally involves contacting the sample with an anti- chimeric fHBP antibody (e.g., an anti-chimeric fHBP mAb, such as a JAR 5 mAb or other appropriate JAR mAb described herein), washing to remove non- specifically bound material, and eluting specifically bound chimeric fHBP. Isolated chimeric fHBP can be further purified by dialysis and other methods normally employed in protein purification methods. In one embodiment, the chimeric fHBP can be isolated using metal chelate chromatography methods.
Host cells
[00149] Any of a number of suitable host cells can be used in the production of chimeric fHBP. In general, the chimeric fHBP described herein may be expressed in prokaryotes or eukaryotes, usually bacteria, more usually E. coli or Neisseria (e.g., N. meningitidis) in accordance with conventional techniques. Thus, the present disclosure further provides a genetically modified host cell, which contains a nucleic acid encoding a chimeric fHBP. Host cells for production (including large scale production) of a chimeric fHBP can be selected from any of a variety of available host cells. Exemplary host cells for expression include those of a prokaryotic or eukaryotic unicellular organism, such as bacteria (e.g., Escherichia coli strains), yeast (e.g., S. cerevisiae, Pichia spp., and the like)., and may include host cells originally derived from a higher organism such as insects, vertebrates, particularly mammals, (e.g. CHO, HEK, and the like). Generally bacterial host cells and yeast are of particular interest for chimeric fHBP production.
[00150] Chimeric fHBPs can be prepared in substantially pure or substantially isolated form (i.e., substantially free from other Νeisserial or host cell polypeptides) or substantially isolated form. In certain embodiments, the chimeric fHBP is present in a composition that is enriched for the polypeptide relative to other components that may be present (e.g., other polypeptides or other host cell components). Purified chimeric fHBP can be provided such that the polypeptide is present in a composition that is substantially free of other expressed polypeptides, e.g., less than 90%, usually less than 60% and more usually less than 50% of the composition is made up of other expressed polypeptides.
Host cells for vesicle production
[00151] Where a chimeric fHBP is to be provided in a membrane vesicle (as discussed in more detail below), a Νeisserial host cell is genetically modified to express a chimeric fHBP. Any of a variety of Neisseria spp. strains can be modified to produce a chimeric fHBP, and, optionally, which produce or can be modified to produce other antigens of interest, such as PorA, can be used in the methods disclosed herein.
[00152] Methods and vectors to provide for genetic modification of Νeisserial strains and expression of a desired polypeptide are known in the art. Exemplary vectors and methods are provided in WO 02/09746 and O'Dwyer et al. Infect Immun 2004;72:6511-80. Strong promoters, particularly constitutive strong promoters are of particular interest. Exemplary promoters include the promoters of porA, porB, lbpB, tbpB, pi 10, hpuAB, lgtF, opa, pi 10, 1st, hpuAB. and rmp.
[00153] Pathogenic Neisseria spp. or strains derived from pathogenic Neisseria spp., particularly strains pathogenic for humans or derived from strains pathogenic or commensal for humans, are of particular interest for use in membrane vesicle production. Exemplary Neisserial spp. include N. meningitidis, N. flavescens, N. gonorrhoeae, N. lactamica, N. polysaccharea, N. cinerea, N. mucosa, N. subflava, N. sicca, N. elongata, and the like. [00154] N. meningitidis strains are of particular interest for genetic modification to express a chimeric fHBP and for use in vesicle production. The strain used for vesicle production can be selected according to a number of different characteristics that may be desired. For example, the strain may be selected according to: a desired PorA type (a "serosubtype", as described above), capsular group, serotype, and the like; decreased capsular polysaccharide production; and the like. For example, the production strain can produce any desired PorA polypeptide, and may express one or more PorA polypeptides (either naturally or due to genetic engineering). Exemplary strains includes those that produce a PorA polypeptide which confers a serosubtype of Pl.7,16; Pl.19,15; Pl.7,1; Pl.5,2; P1.22a,14; Pl.14 ; Pl.5, 10; Pl.7,4; Pl.12,13; as well as variants of such PorA polypeptides which may or may not retain reactivity with conventional serologic reagents used in serosubtyping. Also of interest are PorA polypeptides characterized according to PorA variable region (VR) typing (see, e.g., Russell et al. Emerging Infect Dis 2004 10:674-678; Sacchi CT, et al, Clin Diagn Lab Immunol 1998;5:845-55; Sacchi et al, J. Infect Dis 2000;182:1169-1176). A substantial number of distinct VR types have been identified, which can be classified into VRl and VR2 family "prototypes". A web-accessible database describing this nomenclature and its relationship to previous typing schemes is found at neisseria.org/nm/typing/pora. Alignments of exemplary PorA VRl and VR2 types is provided in Russell et al. Emerging Infect Dis 2004 10:674-678.
[00155] Alternatively or in addition, the production strain can be a capsule deficient strain. Capsule deficient strains can provide vesicle-based vaccines that provide for a reduced risk of eliciting a significant autoantibody response in a subject to whom the vaccine is administered (e.g., due to production of antibodies that cross-react with sialic acid on host cell surfaces). "Capsule deficient" or "deficient in capsular polysaccharide" as used herein refers to a level of capsular polysaccharide on the bacterial surface that is lower than that of a naturally-occurring strain or, where the strain is genetically modified, is lower than that of a parental strain from which the capsule deficient strain is derived. A capsule deficient strain includes strains that are decreased in surface capsular polysaccharide production by at least 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, 90% or more, and includes strains in which capsular polysaccharide is not detectable on the bacterial surface (e.g., by whole cell ELISA using an anti-capsular polysaccharide antibody).
[00156] Capsule deficient strains include those that are capsule deficient due to a naturally-occurring or recombinantly-generated genetic modification. Naturally-occurring capsule deficient strains (see, e.g., Dolan-Livengood et al. J. Infect. Dis. (2003) 187(10): 1616-28), as well as methods of identifying and/or generating capsule-deficient strains (see, e.g., Fisseha et al. (2005) Infect. Immun. 73(7):4070-4080; Stephens et al. (1991) Infect Immun 59(l l):4097-102; Frosch et al. (1990) MoI Microbiol. 1990 4(7): 1215-1218) are known in the art.
[00157] Modification of a Neisserial host cell to provide for decreased production of capsular polysaccharide may include modification of one or more genes involved in capsule synthesis, where the modification provides for, for example, decreased levels of capsular polysaccharide relative to a parent cell prior to modification. Such genetic modifications can include changes in nucleotide and/or amino acid sequences in one or more capsule biosynthesis genes rendering the strain capsule deficient (e.g., due to one or more insertions, deletions, substitutions, and the like in one or more capsule biosynthesis genes). Capsule deficient strains can lack or be non- functional for one or more capsule genes. [00158] Of particular interest are strains that are deficient in sialic acid biosynthesis.
Such strains can provide for production of vesicles that have reduced risk of eliciting anti- sialic acid antibodies that cross-react with human sialic acid antigens, and can further provide for improved manufacturing safety. Strains having a defect in sialic acid biosynthesis (due to either a naturally occurring modification or an engineered modification) can be defective in any of a number of different genes in the sialic acid biosynthetic pathway. Of particular interest are strains that are defective in a gene product encoded by the N-acetylglucosamine- 6-phosphate 2-epimerase gene (known as synX AAF40537.1 or siaA AAA20475), with strains having this gene inactivated being of especial interest. For example, in one embodiment, a capsule deficient strain is generated by disrupting production of a functional synX gene product (see, e.g., Swartley et al. (1994) J Bacterid. 176(5): 1530-4). [00159] Capsule-deficient strains can also be generated from naturally-occurring strains using non-recombinant techniques, e.g., by use of bactericidal anti-capsular antibodies to select for strains that reduced in capsular polysaccharide.
[00160] Where the disclosure involves use of two or more strains (e.g., to produce antigenic compositions containing a chimeric fHBP-presenting vesicles from different strains), the strains can be selected so as to differ in one or more strain characteristics, e.g., to provide for vesicles that differ in the chimeric fHBP used, PorA, and the like.
Preparation Of Vesicles
[00161] The antigenic compositions contemplated by the present disclosure generally include vesicles prepared from Neisserial cells that express a chimeric fHBP. As referred to herein "vesicles" is meant to encompass outer membrane vesicles as well as microvesicles (which are also referred to as blebs).
[00162] In one embodiment, the antigenic composition comprises outer membrane vesicles (OMV) prepared from the outer membrane of a cultured strain of Neisseria meningitidis spp. genetically modified to express a chimeric fHBP. OMVs may be obtained from Neisseria meningitidis grown in broth or solid medium culture, preferably by separating the bacterial cells from the culture medium (e.g. by filtration or by a low-speed centrifugation that pellets the cells, or the like), lysing the cells (e.g. by addition of detergent, osmotic shock, sonication, cavitation, homogenization, or the like) and separating an outer membrane fraction from cytoplasmic molecules (e.g. by filtration; or by differential precipitation or aggregation of outer membranes and/or outer membrane vesicles, or by affinity separation methods using ligands that specifically recognize outer membrane molecules; or by a highspeed centrifugation that pellets outer membranes and/or outer membrane vesicles, or the like); outer membrane fractions may be used to produce OMVs.
[00163] In another embodiment, the antigenic composition comprises microvesicles
(MV) (or "blebs") containing chimeric fHBP, where the MV or blebs are released during culture of a Neisseria meningitidis strain genetically modified to express a chimeric fHBP. For example, MVs may be obtained by culturing a strain of Neisseria meningitidis in broth culture medium, separating whole cells from the broth culture medium (e.g. by filtration, or by a low-speed centrifugation that pellets only the cells and not the smaller blebs, or the like), and then collecting the MVs that are present in the cell-free culture medium (e.g. by filtration, differential precipitation or aggregation of MVs, or by a high-speed centrifugation that pellets the blebs, or the like). Strains for use in production of MVs can generally be selected on the basis of the amount of blebs produced in culture (e.g., bacteria can be cultured in a reasonable number to provide for production of blebs suitable for isolation and administration in the methods described herein). An exemplary strain that produces high levels of blebs is described in PCT Publication No. WO 01/34642. In addition to bleb production, strains for use in MV production may also be selected on the basis of NspA production, where strains that produce higher levels of NspA may be of particular interest (for examples of N. meningitidis strains having different ΝspA production levels, see, e.g., Moe et al. (1999 Infect. Immun. 67: 5664). Other strains of interest for use in production of blebs include strains having an inactivated GΝ33 gene, which encodes a lipoprotein required for cell separation, membrane architecture and virulence (see, e.g., Adu-Bobie et al. Infect Immun. 2004;72:1914-1919).
[00164] The antigenic compositions of the present disclosure can comprise vesicles from one strain, or from 2, 3, 4, 5 or more strains, which strains may be homologous or heterologous, usually heterologous, to one another. For example, the strains may be homologous or heterologous with respect to PorA. In one embodiment, the vesicles can be prepared from strains that express more than one chimeric fHBP (e.g., 1, 2, 3, or more chimeric fHBP) which may be composed of fHBP amino acid sequences from different variants (v.l, v.2, or v.3) or subvariants (e.g., a subvariant of v.l, v.2, or v.3). [00165] The antigenic compositions can comprise a mixture of OMVs and MVs presenting the same or different chimeric fHBPs, where the chimeric fHBPs may optionally present epitopes from different combinations of fHBP variants and/or subvariants and where the OMVs and/or MVs may be from the same or different strains. Vesicles from different strains can be administered as a mixture, or can be administered serially. [00166] Where desired (e.g., where the strains used to produce vesicles are associated with endotoxin or particular high levels of endotoxin), the vesicles are optionally treated to reduce endotoxin, e.g., to reduce toxicity following administration. Although less desirable as discussed below, reduction of endotoxin can be accomplished by extraction with a suitable detergent (for example, BRIJ-96, sodium deoxycholate, sodium lauroylsarcosinate, Empigen BB, Triton X-IOO, TWEEN 20 (sorbitan monolaurate polyoxyethylene), TWEEN 80, at a concentration of 0.1-10%, preferably 0.5-2%, and SDS). Where detergent extraction is used, it is preferable to use a detergent other than deoxycholate.
[00167] In some embodiments the vesicles of the antigenic compositions are prepared without detergent, e.g., without use of deoxycholate. Although detergent treatment is useful to remove endotoxin activity, it may deplete the native fHBP lipoprotein and/or chimeric fHBP (including lipidated chimeric fHBP) by extraction during vesicle production. Thus it may be particularly desirable to decrease endotoxin activity using technology that does not require a detergent. In one approach, strains that are relatively low producers of endotoxin (lipopolysaccharide, LPS) are used so as to avoid the need to remove endotoxin from the final preparation prior to use in humans. For example, the vesicles can be prepared from Neisseria mutants in which lipooligosaccharide or other antigens that may be undesirable in a vaccine (e.g. Rmp) is reduced or eliminated.
[00168] Vesicles can be prepared from N. meningitidis strains that contain genetic modifications that result in decreased or no detectable toxic activity of lipid A. For example, such strain can be genetically modified in lipid A biosynthesis (Steeghs et al. Infect Immun 1999;67:4988-93; van der Ley et al. Infect Immun 2001;69:5981-90; Steeghs et al. J Endotoxin Res 2004; 10: 113-9; Fissha et al, Infect Immun 73:4070,.2005). The immunogenic compositions may be detoxified by modification of LPS, such as downregulation and/or inactivation of the enzymes encoded by lpxLl or lpxL2, respectively. Production of a penta- acylated lipid A made in lpxLl mutants indicates that the enzyme encoded by lpxLl adds the C12 to the Ν-linked 3-OH C14 at the 2' position of GlcΝ II. The major lipid A species found in lpxL2 mutants is tetra-acylated, indicating the enzyme encoded by lpxL2 adds the other C12, i.e., to the Ν-linked 3-OH C14 at the 2 position of GlcΝ I. Mutations resulting in a decreased (or no) expression of these genes (or decreased or no activity of the products of these genes) result in altered toxic activity of lipid A (van der Ley et al. 2001 ;69:5981-90). Tetra-acylated (lpxL2 mutant) and penta acylated (lpxLl mutant) lipid A are less toxic than the wild-type lipid A. Mutations in the lipid A 4' -kinase encoding gene (lpxK) also decreases the toxic activity of lipid A. Of particular interest for use in production of vesicles (e.g., MV or OMV) are N. meningitidis strains genetically modified so as to provide for decreased or no detectable functional LpxLl -encoded protein. Such vesicles provide for reduced toxicity as compared to N. meningitidis strains that are wild-type for LPS production, while retaining immunogenicity of chimeric fHBP.
[00169] LPS toxic activity can also be altered by introducing mutations in genes/loci involved in polymyxin B resistance (such resistance has been correlated with addition of aminoarabinose on the 4' phosphate of lipid A). These genes/loci could be pmrE that encodes a UDP-glucose dehydrogenase, or a region of antimicrobial peptide-resistance genes common to many enterobacteriaciae which could be involved in aminoarabinose synthesis and transfer. The gene pmrF that is present in this region encodes a dolicol-phosphate manosyl transferase (Gunn J. S., Kheng, B. L., Krueger J., Kim K., Guo L., Hackett M., Miller S. I. 1998. MoI. Microbiol. 27: 1171-1182).
[00170] Mutations in the PhoP-PhoQ regulatory system, which is a phospho-relay two component regulatory system (e.g., PhoP constitutive phenotype, PhoPc), or low Mg++ environmental or culture conditions (that activate the PhoP-PhoQ regulatory system) lead to the addition of aminoarabinose on the 4' -phosphate and 2-hydroxymyristate replacing myristate (hydroxy lation of myristate). This modified lipid A displays reduced ability to stimulate E-selectin expression by human endothelial cells and TNF secretion from human monocytes.
[00171] Polymyxin B resistant strains are also suitable for use, as such strains have been shown to have reduced LPS toxicity (see, e.g., van der Ley et al. 1994. In: Proceedings of the ninth international pathogenic Neisseria conference. The Guildhall, Winchester, England). Alternatively, synthetic peptides that mimic the binding activity of polymyxin B may be added to the antigenic compositions to reduce LPS toxic activity (see, e.g., Rustici et al. 1993, Science 259:361-365; Porro et al. Prog Clin Biol Res. 1998;397:315-25). [00172] Endotoxin can also be reduced through selection of culture conditions. For example, culturing the strain in a growth medium containing 0.1 mg-100 mg of aminoarabinose per liter medium provides for reduced lipid toxicity (see, e.g., WO 02/097646).
FORMULATIONS
[00173] "Antigen composition", "antigenic composition" or "immunogenic composition" is used herein as a matter of convenience to refer generically to compositions comprising a chimeric fHBP as disclosed herein, which chimeric fHBP may be optionally conjugated to further enhance immunogenicity. Compositions useful for eliciting antibodies, particularly antibodies against Neisseria meningitidis group B (NmB), in a human are specifically contemplated by the present disclosure. Antigenic compositions can contain 2 or more different chimeric fHBPs, where the chimeric fHBPs may present epitopes from different combinations of fHBP variants and/or subvariants.
[00174] Antigenic compositions generally comprise an immunologically effective amount of chimeric fHBP, and may further include other compatible components, as needed. By "immunologically effective amount" is meant that the administration of that amount to an individual, either in a single dose, as part of a series of the same or different antigenic compositions, is effective to elicit an antibody response effective for treatment or prevention of a symptom of, or disease caused by, for example, infection by Neisseria, particularly N. meningitidis, more particularly Group B N. meningitidis. This amount varies depending upon the health and physical condition of the individual to be treated, age, the capacity of the individual's immune system to produce antibodies, the degree of protection desired, the formulation of the vaccine, the treating clinician's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
[00175] Dosage regimen may be a single dose schedule or a multiple dose schedule
(e.g., including booster doses) with a unit dosage form of the antigenic composition administered at different times. The term "unit dosage form," as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of the antigenic compositions of the present invention in an amount sufficient to produce the desired effect, which compositions are provided in association with a pharmaceutically acceptable excipient (e.g., pharmaceutically acceptable diluent, carrier or vehicle). The antigenic composition may be administered in conjunction with other immunoregulatory agents.
[00176] Antigenic compositions can be provided in a pharmaceutically acceptable excipient, which can be a solution such as a sterile aqueous solution, often a saline solution, or they can be provided in powder form. Such excipients can be substantially inert, if desired. [00177] The antigenic compositions can further comprise an adjuvant. Examples of known suitable adjuvants that can be used in humans include, but are not necessarily limited to, alum, aluminum phosphate, aluminum hydroxide, MF59 (4.3% w/v squalene, 0.5% w/v Tween 80™, 0.5% w/v Span 85), CpG-containing nucleic acid (where the cytosine is unmethylated), QS21, MPL, 3DMPL, extracts from Aquilla, ISCOMS, LT/CT mutants, poly(D,L-lactide-co-glycolide) (PLG) microparticles, Quil A, interleukins, and the like. For experimental animals, one can use Freund's, Ν-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), Ν-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor- MDP), Ν-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(l'-2'-dip- almitoyl-sn- glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion. The effectiveness of an adjuvant may be determined by measuring the amount of antibodies directed against the immunogenic antigen or antigenic epitope thereof. [00178] Further exemplary adjuvants to enhance effectiveness of the composition include, but are not limited to: (1) oil-in- water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59™ (WO 90/14837; Chapter 10 in Vaccine design: the subunit and adjuvant approach, eds. Powell & Newman, Plenum Press 1995), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing MTP-PE) formulated into submicron particles using a microfluidizer, (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic -blocked polymer L121, and thr-MDP either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) RIBI ™ adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components such as monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox ™); (2) saponin adjuvants, such as QS21 or Stimulon ™(Cambridge Bioscience, Worcester, Mass.) may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes), which ISCOMS may be devoid of additional detergent e.g WO 00/07621; (3) Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (4) cytokines, such as interleukins (e.g. IL-I, IL-2, IL- 4, IL-5, IL-6, IL-7, IL- 12 (WO99/44636), etc.), interferons (e.g. gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; (5) monophosphoryl lipid A (MPL) or 3-O-deacylated MPL (3dMPL) e.g. GB-2220221, EP-A- 0689454, optionally in the substantial absence of alum when used with pneumococcal saccharides e.g. WO 00/56358; (6) combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulsions e.g. EP-A-0835318, EP-A-0735898, EP-A-0761231; (7) oligonucleotides comprising CpG motifs (Krieg Vaccine 2000, 19, 618-622; Krieg Curr opin MoI Ther2001 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., 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, where the cytosine is unmethylated; (8) a polyoxyethylene ether or a polyoxyethylene ester e.g WO 99/52549; (9) a polyoxyethylene sorbitan ester surfactant in combination with an octoxynol (WO 01/21207) or a polyoxyethylene alkyl ether or ester surfactant in combination with at least one additional non-ionic surfactant such as an octoxynol (WO 01/21152); (10) a saponin and an immunostimulatory oligonucleotide (e.g. a CpG oligonucleotide) (WO 00/62800); (11) an immunostimulant and a particle of metal salt e.g WO 00/23105; (12) a saponin and an oil-in- water emulsion e.g. WO 99/11241; (13) a saponin (e.g QS21)+3dMPL+IM2 (optionally+a sterol) e.g WO 98/57659; (14) other substances that act as immunostimulating agents to enhance the efficacy of the composition. Muramyl peptides include N-acetyl-muramyl-L- threonyl-D-isoglutamine (thr-MDP), N-25 acetyl-normuramyl-L-alanyl-D-isoglutamine (nor- MDP), N-acetylmuramyl-L-alanyl-D-isoglutarninyl-L-alanine-2-(l'-2'-dipalmitoyl— sn- glycero-3-hydroxyphosphoryloxy)-ethylamine MTP-PE), etc. Adjuvants suitable for administration to a human are of particular interest.
[00179] The antigen compositions may comprise other components, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium, carbonate, and the like. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
[00180] The concentration of chimeric fHBP in a formulation can vary widely (e.g., from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight) and will usually be selected primarily based on fluid volumes, viscosities, and patient-based factors in accordance with the particular mode of administration selected and the patient's needs.
[00181] Chimeric fHBP-containing formulations can be provided in the form of a solution, suspension, tablet, pill, capsule, powder, gel, cream, lotion, ointment, aerosol or the like. It is recognized that oral administration can require protection of the compositions from digestion. This is typically accomplished either by association of the composition with an agent that renders it resistant to acidic and enzymatic hydrolysis or by packaging the composition in an appropriately resistant carrier. Means of protecting from digestion are well known in the art. [00182] Chimeric fHBP-containing formulations can also provided so as to enhance serum half-life of chimeric fHBP following administration. For example, where isolated chimeric fHBP are formulated for injection, the chimeric fHBP may be provided in a liposome formulation, prepared as a colloid, or other conventional techniques for extending serum half-life. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728 and 4,837,028. The preparations may also be provided in controlled release or slow-release forms.
IMMUNIZATION
[00183] The chimeric fHBP-containing antigenic compositions are generally administered to a human subject that is at risk of acquiring a Neisserial disease so as to prevent or at least partially arrest the development of disease and its complications. An amount adequate to accomplish this is defined as a "therapeutically effective dose." Amounts effective for therapeutic use will depend on, e.g., the antigenic composition, the manner of administration, the weight and general state of health of the patient, and the judgment of the prescribing physician. Single or multiple doses of the antigenic compositions may be administered depending on the dosage and frequency required and tolerated by the patient, and route of administration.
[00184] The chimeric fHBP-containing antigenic compositions are generally administered in an amount effective to elicit an immune response, particularly a humoral immune response, in the host. As noted above, amounts for immunization will vary, and can generally range from about 1 μg to 100 μg per 70 kg patient, usually 5 μg to 50 μg/70 kg. Substantially higher dosages (e.g. 10 mg to 100 mg or more) may be suitable in oral, nasal, or topical administration routes. The initial administration can be followed by booster immunization of the same of different chimeric fHBP-containing antigenic composition. Usually vaccination involves at least one booster, more usually two boosters. [00185] In general immunization can be accomplished by administration by any suitable route, including administration of the composition orally, nasally, nasopharyngeally, parenterally, enterically, gastrically, topically, transdermally, subcutaneously, intramuscularly, in tablet, solid, powdered, liquid, aerosol form, locally or systemically, with or without added excipients. Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa. (1980).
[00186] An anti-chimeric fHBP immune response can be assessed by known methods
(e.g. by obtaining serum from the individual before and after the initial immunization, and demonstrating a change in the individual's immune status, for example an immunoprecipitation assay, or an ELISA, or a bactericidal assay, or a Western blot, or flow cytometric assay, or the like).
[00187] In one embodiment, the antigenic compositions can be administered to a human subject that is immunologically naive with respect to Neisseria meningitidis. In a particular embodiment, the subject is a human child about five years or younger, and preferably about two years old or younger, and the antigenic compositions are administered at any one or more of the following times: two weeks, one month, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months, or one year or 15, 18, or 21 months after birth, or at 2, 3, 4, or 5 years of age. [00188] It may be generally desirable to initiate immunization prior to the first sign of disease symptoms, or at the first sign of possible or actual exposure to infection or disease (e.g., due to exposure or infection by Neisseria).
EXAMPLES
[00189] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
MATERIALS AND METHODS
[00190] The following methods and materials were used in the Examples below.
[00191] Gene cloning. Wild-type fHBP genes were amplified from genomic DNA by
PCR and cloned into pGEM-T-Easy (Promega). The resulting plasmids were treated with the restriction enzymes Ndel and Xhol and the approximately 800 base-pair fragments containing the fHBP coding sequences were ligated into pET21b (Νovagen) cut with the same two enzymes. The plasmid clones were confirmed by DN A sequence determination of PCR products obtained by the amplification of the plasmid with primers specific for the T7 promotor and terminator regions. The plasmids encoded the full-length fHBP proteins except for the amino-terminal 19 amino acid signal sequence and 7 presumably flexible N-terminal residues, and included a C-terminal hexa-histidine (HiS6) tag originating from the pET21b plasmid.
[00192] fHBP Chimera I lacking a signal sequence was constructed by PCR amplification of the region encoding residues 8-135 from genomic DNA from strain MC58 (fHBP v.l) and that encoding residues 136-255 from strain 8047 (v.2). The two fragments, including an overlapping region of 48 base pairs centered around amino acids 136-139, were assembled by PCR amplification using external, gene-specific primers containing the Ndel and Xhol restriction sites. The chimeric gene was cloned into pET21b as described for wild- type genes above.
[00193] Site-specific mutagenesis. Site-specific mutagenesis was used to test predictions of amino acid residues involved in anti-fHBP mAb epitopes and to create Chimera II. Mutagenesis was performed using the QuikChange II kit (Stratagene) using 10 ng of plasmid template and the manufacturer's protocols. For testing residues putatively involved in mAb epitopes, mutagenesis reactions were performed on pET21 -based plasmids encoding wild-type fHBP genes from various strains. A residue in a reactive sequence was changed to that naturally present in a non-reactive sequence or vice versa. To construct Chimera II, site-specific mutagenesis was used to introduce the A174K substitution into the pET21 plasmid encoding Chimera I. Mutant fHBP genes were confirmed by DN A sequencing as described for the wild-type plasmids, above.
[00194] fHBP expression and purification. Wild-type, mutant and chimeric fHBPs were expressed in Escherichia coli BL21(DE3) (Novagen). fHBP purifications were performed from 1 L cultures. Mid-exponential cultures (optical density at 600 nm of 0.5-0.6) were grown at 37 0C, induced with 0.5 mM isopropylthiogalactoside for 3-4 h and the bacteria harvested by centrifugation. The cells were lysed by incubation with chicken egg white lysozyme (Sigma) and two freeze/thaw cycles. Bacterial lysates were treated with DNase and RNase (Sigma) and protease inhibitors (Complete EDTA-free, Roche) and clarified by centrifugation at 13,000 x g. Recombinant fHBPs were purified by nickel chelate chromatography using Ni-NTA agarose (Qiagen) and buffers recommended by the supplier. Fractions containing purified fHBP were pooled and dialyzed against PBS (Roche) containing 5% (w/v) sucrose and 0.01% NaN3, filter sterilized and stored at 4 0C. [00195] mAb preparation. We generated hybridoma cell lines from spleens from CD-I mice immunized with recombinant fHBP variant group 2 (gene from strain 2996) or variant group 3 (gene from strain M1239) using methods previously described for preparation of hybridoma cell lines secreting mAbs against fHBP v.l protein (Welsch et al., J. Immunol. 2004). mAbs were precipitated from tissue culture supernatants with 50% saturated (NH4^SO4 and dialyzed against phosphate-buffered saline (PBS; Roche). IgG isotypes were determined using Clonotyping-AP reagents (Southern Biotech).
[00196] Direct-binding and inhibition ELISA. Binding of the mAbs to recombinant fHBP was measured by ELISA. The wells of a microtiter plate (Immulon 2B; Thermo Electron Corp.) were coated with 1 μg/ml of recombinant fHBP in PBS and incubated overnight at 4 0C. The plates were blocked with PBS containing 0.1% Tween-20 (Sigma) (PBST) and 1% BSA (Sigma). The primary antibodies were anti-fHBP mAbs (0.016 to 5 μg/ml) and the secondary antibody was rabbit anti-mouse IgG-alkaline phosphatase (Zymed; 1:5,000), each diluted in PBST. After 1 h at room temperature, alkaline phosphatase substrate (Sigma) was added and the absorbance at 405 nm was measured after 30 min. [00197] For competitive inhibition ELISAs, one mAb was held at a fixed concentration sufficient to obtain an OD at 405 nm of 1.0 determined by direct binding ELISA, as described above. A second mAb of a different isotype was added together with the first mAb to the wells of a microtiter plate coated with the antigen as described above, at concentrations ranging from 0.4 to 50 μg/ml. The secondary mAb was an isotype specific mouse anti-IgG- alkaline phosphatase conjugate. The ELISA was developed as described above. [00198] Inhibition of binding of factor H. The ability of an anti-fHBP mAb to inhibit binding of fH to fHBP was measured by ELISA. Wells of a microtiter plate were coated with rfHBP as described above. Dilutions containing 0.016 to 50 μg/ml of the mAb were added to the wells together with 50 μg/ml purified fH (Complement Technology, Inc.). The plates were incubated overnight at 40C. Bound fH was detected with goat polyclonal anti-fH (Bethyl Laboratories) (1: 1000) followed by mouse anti-goat IgG alkaline phosphatase conjugate (Santa Cruz Biotech) (1:2000). Both steps were performed at room temperature for 2 hours. After washing, substrate was added and developed as described above for the antibody binding ELISA.
[00199] Western blotting. One mL of bacterial culture was grown and induced as described above (see fHBP expression and purification, above). The cells were harvested by centrifugation and were resuspended in 0.5 mL of Ix LDS sample buffer (Invitrogen) containing 25 mM 2-ME.Bacterial lysates were separated by SDS-PAGE using 4-12% NuPAGE polyacrylamide gels and MES SDS-PAGE buffer (Invitrogen). Proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Millipore). The membranes were blocked using PBST containing 2% nonfat dry milk (Carnation, Nestle, Inc.). The membranes were washed, incubated with the different anti-fHBP mAbs (1 to 5 μg/ml) or, as a control for protein expression by the different clones, 0.02 μg/ml of Penta-His mAb (Qiagen). The membranes were washed in PBST and incubated with a 1:10,000 dilution of a rabbit anti-mouse IgG-horseradish peroxidase conjugate (Zymed) and washed again. The membranes were developed with a chemiluminescent substrate (ECL+; GE Healthcare) and visualized on a Storm 840 imager (Molecular Dynamics).
[00200] Mouse immunization. Groups (5 mice each) of five-week old CD-I mice
(Charles River) were immunized with four doses containing 20 μg of wild-type or chimeric fHBP vaccines or adjuvant alone at two-week intervals. Each experimental or control vaccine was administered with aluminum hydroxide or Freund's adjuvant (FA) (complete FA for the first dose and incomplete FA for subsequent doses).
[00201] Bactericidal activity. Complement-mediated bactericidal activity was measured as described previously using washed, log-phase bacteria grown in Mueller-Hinton broth supplemented with 0.25 % glucose and 0.02 mM CMP-NANA to an OD620 of 0.6. The buffer was Dulbecco's phosphate buffered saline (Mediatech, Inc.) containing 0.9 mM CaCl2 x 2 H2O, 0.5 mM MgCl2 x 6 H2O and 1% (w/v) BSA. The complement source was human serum from a healthy adult with no detectable intrinsic bactericidal activity. For synergism of mAb bactericidal activity, equal quantities of two mAbs ranging from 0.4 to 50 ug/ml (final concentration) were used. The bactericidal activity (BC50) of the mouse antiserum (or mAb combination) was defined by the dilution (or mAb concentration) that gave a 50% decrease in the number of CFU after 60 min incubation at 37 0C as compared with the CFU at time 0 in the negative control reactions.
OVERVIEW OF EXAMPLES
[00202] This study used a panel of twelve murine monoclonal antibodies (mAbs) that had been prepared against recombinant proteins representative of the three major variant groups of factor H binding protein (fHBP). These variant groups are designated variant 1 (v.l; Welsch et al. J Immunol 2004; 172: 5606-15), variant 2 (v.2, Beernink et al. J Infect Dis 2007; 195: 1472-9) and v.3 (Beernink et al, (2008) Infect Immun 76: 4232-4240). As illustrated by the summary of selected features of these mAbs in the tables of Figures 17 and 18, each of these mAbs is bactericidal when combined with a second mAb. In addition JAR 1 and JAR 3 are individually bactericidal when tested against certain strains expressing v.l fHBP. [00203] The results summarized in Figure 18 are evidence that the epitopes recognized by each of the mAbs is surface-exposed on encapsulated meningococcal strains and capable of interacting with protective antibodies.
[00204] Nine of these JAR mAbs were used as tools to determine amino acid residues in fHBP that contribute to the epitopes for binding of these mAbs so as to provide for structural predictions on the locations of epitopes of fHBP from different variant groups that interact with bactericidal antibodies. This information could then be used to construct chimeric fHBP vaccines that express epitopes from more than one variant group and that are capable of eliciting antibodies that confer protection against strains expressing different fHBP variants.
[00205] For convenience in facilitating prior studies aimed at identifying bactericidal regions of fHBP, the protein was divided into three domains, designated A, B and C (Giuliani et all. Infect Immun 2005 ;73: 1151-60). As discussed above, the A domain is highly conserved across v.l and v.2 variant groups, whereas the B and C domains contain sequences that diverge among strains. Previously, the only known fHBP epitope interacting with a bactericidal mAb was located in the C domain at R204 (Giuliani et al. supra ). (Note that the convention of numbering amino acid residues beginning with the first residue after the signal sequence is adopted herein). However, a chimeric protein vaccine composed of the B domain from a variant 3 strain (B3) fused with the C domain of a variant 1 strain (Ci) failed to elicit protective bactericidal responses against strains with either variant 1 or 3 fHBP. [00206] As will be set out in more detail below, the inventors identified the location of an epitope defined by two anti-fHBP mAbs, JAR 3 and 5 (referred to herein as the "JAR3/5" epitope), in the Bi domain around residue G121. JAR 3 individually was known to be bactericidal with human complement against strains from sequence type (ST) complex 32 (Welsch et al. J Immunol 2004;172:5606-15) and, both JAR 3 and JAR 5 when combined with other mAbs were broadly bactericidal against strains expressing sub variants of variant 1 fHBP (Welsch et al., supra ). JAR 3 (Madico et al. J Immunol 2006;177:501-10) and JAR 5 also inhibited binding of factor H (fH) to the surface of encapsulated N. meningitidis strains. It should be noted that with the exception of JAR3/5, all of the other mAbs mapped to date were found to bind to epitopes that are part of the C domain.
[00207] The observations relating to the JAR 3/5 epitope being present in the B domain formed the rationale for fusing the portion of the protein of v.1 B domain containing the JAR3/5 epitope (e.g., up to residue 136) with the remaining portion of the B domain and entire C domain of the v.2 protein (Chimera I). Inclusion of the JAR 3/5 epitope from v.1 at resides near and including G121 elicited broadly protective antibodies to v.l and v.l subvariant strains.
[00208] Further, by inclusion of the other epitopes such as JAR 10, 11 and 13, the chimeric fHBP provided for a polypeptide that elicited broadly protective antibodies against v.2 or v.3 strains. When mice were immunized with either Chimera I or Chimera II vaccines, the animals developed serum bactericidal antibody responses against strains expressing fHBP v.l, v.2 or v.3 whereas as expected the serum bactericidal antibody responses of the control mice immunized with the wild-type recombinant fHBP v.l were nearly entirely restricted to strains expressing fHBP v.l , and those of the control mice immunized with the wild-type recombinant fHBP v.2 were restricted to v.2 or v.3 (which from previous studies were known to cross-react (Masignani 2007 J Exp Med, supra and Beernink 2007 J. Infect Dis supra ). The results provide proof of concept that an individual chimeric protein can elicit antibodies that are bactericidal with human complement against strains expressing fHBP from different variant groups.
[00209] Thus, in contrast to the B3C1 chimeric previously reported, a chimeric protein vaccine composed of the A1 domain (which is highly conserved across variant 1 and 2), the N-terminal portion of the B1 domain expressing the JAR 3/5 epitope fused with the distal terminal portion of the B1 domain and C1 domain elicited cross-protective bactericidal antibodies in immunized mice.
[00210] The JAR 11 epitope is expressed by about one-third of disease-producing N. meningitidis strains in the U.S. that express fHBP v.2 or v.3 (Beernink 2007 J. Infect Dis, supra). Approximately 50 percent of the JAR 11-negative strains with v.2 or v.3 fHBP express the JAR 32/35 epitope. Therefore, to increase coverage against these strains, the Chimera II vaccine was prepared, in which a single amino acid change, A174K, was introduced into the C domain that inactivated the epitope recognized by JAR 11 and introduced the epitope recognized by JAR 32/35. Despite engineering expression of the JAR 11 epitope in Chimera I and the JAR 32 epitope in Chimera II, there was no statistically significant differences in the respective serum bactericidal antibody responses of mice immunized either vaccine against strains expressing v.2 or v.3 fHBP that were JAR 11- positive or JAR 32-positive.
[00211] As set out below, it was later found that binding of antibody to an epitope located near residue 174 (i.e., JAR 11 in some strains, and JAR 32 in others; see Figure 1) was not sufficient to elicit significant complement-mediated bactericidal activity in the absence of a second mAb binding to an epitope associated with ion pair at residues 180 and 192 (such as JAR 10 in some strains or JAR 33 in others (See Table in Fig. 20)). Among wild-type strains expressing fHBP v.2 or v.3, expression of JAR 32 is often associated with expression of JAR 33 (for example, strains 03S-0658, M1239 and SK104, Table in Figure 8A), while expression of JAR 11 is usually associated with expression of JAR 10 (see for example our strains 8047, MD1435 and MD1321, Figure 8A). This insight pointed to production of chimeric fHBP vaccines effective against JAR 32-positive strains by also introducing the JAR 33 epitope.
[00212] Further, as set out below, it was discovered that bactericidal activity of antibodies is enhanced when two antibodies bind their respective epitopes located at a distance of about 18 - 20 A. Stated differently, a greater bactericidal effect was observed when two antibodies bound epitopes located about 18-20 A apart on the chimeric fHBP compared to the bactericidal effect of these antibodies alone. In contrast, binding of two antibodies to epitopes positioned at a greater distance apart (e.g., > about 27 A) did not enhance bactericidal activity, which may be due to the reduced ability of these bound antibodies to provide for enhanced interaction with CIq of the complement cascade. Binding of two antibodies to epitopes positioned < about 14 A apart also did not provide for enhanced bactericidal activity, which may be the result of steric hindrance of binding of one antibody by the other. Thus, chimeric fHBP which contain epitopes that elicit antibodies that bind to epitopes within about 18 - 20 A apart can provide for further enhanced bactericidal antibody production. Examples of such epitope pairs are those epitopes bound by JAR 10 and JAR 11; and by JAR 33 and JAR 32/ 35 (JAR 32 and JAR 35 bind to the same (or overlapping) epitopes).
[00213] The effect of chimeric fHBP polypeptides as vaccines can be further enhanced by including epitopes that elicit antibodies that block fH binding. For example, as set out below, when the epitopes bound by JAR 13 (v.2 epitope), JAR 11 (v.2 epitope), and JAR 32/35 (v.3 epitope) are bound by antibody, binding of fHBP to fH is inhibited. Thus, the presence of such fH-binding epitopes in the chimeric fHBP polypeptides can provide for production of antibodies that can facilitate protection through this pathway. [00214] Details of the studies that led to this discovery are set out below. EXAMPLE 1: IDENTIFICATION OF AMINO ACID RESIDUES OF FHBP EPITOPES IMPORTANT FOR JAR 3/5 MAB BINDING
[00215] Selected known properties of JAR mAbs are summarized in the tables of
Figures 17 and 18. Notably, JAR 3 and JAR 5 were known to bind to v.l or subvariants of v.l fHBP expressed by strains MC58, 4243, M1390, and NZ98/254 but not to strain M6190 (Welsch et al. J Immunol 2004;172:5606-15). Further, fHBP expressed by M6190 had an arginine at position 121 (R121) whereas the fHBPs from the four reactive strains had glycine at position 121 (G121) (Welsch et al. J Immunol 2004; 172:5606- 15). [00216] In order to confirm that G121 was important for JAR 3 and JAR 5 binding, site-specific mutagenesis was used to change the glycine residue at position 121 in the fHBP sequence of strain MC58 to arginine.
[00217] As illustrated in the Western blots of Figure 1, the G121R substitution resulted in loss of JAR 3 and JAR 5 reactivity (Figure 1, Panel A). The converse change in fHBP from strain M6190, R121G, introduced the JAR 5 epitope (Figure 1, Panel A, lane 6) and, to a lesser extent, the JAR 3 epitope (Figure 1, Panel B, lane 6). The weaker signals for the M6190 mutant R140G protein, particularly for JAR 3, indicated that additional residues may have been important for the these epitopes. The Penta-His control mAb showed that the wild-type and mutant proteins were produced in similar quantities (Figure 1, Panel C). [00218] Additional evidence that JAR 3 and JAR 5 recognized overlapping epitopes was derived from competitive inhibition experiments.
[00219] The results are shown in Figure 2. JAR 5 (5 μg/ml) inhibited binding of JAR 3 by greater than 90% (Figure 2, Panel A) and the reciprocal reaction with JAR 3 inhibited binding of JAR 5 (Figure 2, Panel B). In contrast, there was no detectable inhibition of binding of JAR 4 by JAR 3 (50 μg/ml) or JAR 5 (50 μg/ml) (Figure 2C). JAR 4 also did not inhibit binding of JAR 3 (Figure 2, Panel A) or JAR 5 (Figure 2, Panel B). The positive control, a 1:10 dilution of rabbit anti-fHBP v.l antiserum, inhibited binding of all three mAbs, whereas pre-immune rabbit serum and negative control mAbs gave less than 7% inhibition. Thus JAR 3 and JAR 5 recognize overlapping (or identical) epitopes, since each of these mAbs inhibited binding of the other to fHBP. EXAMPLE 2: IDENTIFICATION OF AMINO ACID RESIDUES OF FHBP EPITOPES IMPLICATED IN JAR MAB BINDING
[00220] To investigate the epitopes defined by the remaining anti-fHBP mAbs in the panel, site-specific mutagenesis was used to create knock-outs (KO) of recombinant fHBPs. For nine of the mAbs, an fHBP KO lacking the indicated residue resulted in a significant loss of binding of the corresponding JAR mAb as measured by Western blot and/or ELISA (see Table in Figure 29.) For seven of the mAbs, it was demonstrated that the respective mAb that was negative for binding became positive for binding after introduction of one or two of the corresponding amino acid substitutions (see Table in Figure 29). Taken together, one or both of these strategies was successful in identifying amino acid residues involved in the reactivity of nine of the JAR mAbs (Figure 3).
[00221] Figures 30-32 provide supporting data for identification of residues involved in JAR mAb binding. E. coli lysates containing plasmids expressing the respective wild-type and mutant fHBPs were analyzed by Western blot using the appropriate JAR mAb, as well as a control antibody to detect an epitope tag present on the fHBP (penta-His). [00222] Specifically, Figure 30 is a Western blot indicating residues involved in the
JAR 10 and JAR 33 epitopes, in which E. coli lysates containing plasmids expressing the respective wild-type and mutant fHBPs were analyzed by Western blot with JAR 10 (Panel A) or Penta-His mAb (Panel B). Figure 31 is a Western blot indicating a residue involved in the JAR 11, JAR 32 and JAR 35 epitopes, in which E. coli lysates containing plasmids expressing the respective wild-type and mutant fHBPs were analyzed by Western blot with JAR 32 (Panel A), JAR 35 (Panel B), JAR 11 (Panel C) or Penta-His mAb (Panel D). Figure 32 is a Western blot indicating residue involved in the JAR 13 epitope, in which E. coli lysates containing plasmids expressing wild-type and mutant fHBPs were were probed with JAR 13 (Panel A) or Penta-His mAb (Panel B) and anti-mouse IgG-HRP secondary antibody. [00223] It should be noted that the numbering of the amino residues used herein is with reference to the mature protein sequence (i.e. lacking the signal) of fHBP from strain MC58 (i.e., the fHBP amino acid sequence of MC58 was used as the reference v.l fHBP amino acid sequence). Because the total number of amino acid residues in v.2 and/or v.3 fHBPs differ from the total number of amino acid residues in v.l fHBPs, the amino acid sequences of the v.2 protein (with fHBP from strain 8047 used as the reference v.2 amino acid sequence) and v.3 fHBP (with fHBP from strain M1239 used as the reference v.3 amino acid sequence) differ by -1 and +7 amino acid residues, respectively, from that of MC58. Thus, for example, a leucine residue (L) referred to using the numbering system herein as being at position 166 of the v.2 or v.3 fHBP sequence is actually at position 165 of the v.2 protein and at position 173 in the v.3 protein based on the actual amino acid sequence of these proteins, rather than on the numbering used herein based on the alignment of these sequences with v.1 fHBP of MC58.
[00224] In addition, the role of JAR mAb epitopes in fH binding by fHBP was investigated. Figure 19 is a series of graphs showing the ability of representative JAR mAbs prepared against fHBP v.2 (8047), v.3 (MI239), or v.l (MC58) recombinant proteins to give concentration-dependent inhibition of binding of fH to rfHBP v.2 (Panel A), rfHBP v.3 (Panel B), or rfHBP v.l (Panel C) in an ELISA. These data show that some of the contact residues defined by the JAR mAb epitopes are involved in binding to fH. These data thus argue for possible inclusion or preservation of JAR mAb epitopes that are involved in fH binding since blocking of binding of fH to N. meningitidis can provide a further mechanism of protection.
EXAMPLE 3: PRODUCTION OF CHIMERIC FHBP CONTAINING v.l AND V.2 EPITOPES
[00225] As noted above, the epitopes defined by JAR 3 and JAR 5, are located within the B domain of variant 1 fHBP beginning approximately 19 amino acid residues N-terminal to the start of α-helix AH2, or approximately 15 amino acid residues N-terminal to the amino acid sequence of GEHT,. A first chimeric fHBP (referred to herein as "Chimera I") was constructed by combining a portion of the gene encoding the A domain and the N-terminal portion of the B domain up to residue G136 from v.l fHBP of MC58 with the distal portion of the gene encoding the alpha-helix of the B domain and C domain of v.2 fHBP from strain 8047. The residue at position G136 was used as a convenient crossover position (the point at which the chimeric sequence "shifted" from that of the v.l fHBP to that of the v.2 fHBP). G136 is N-terminal the α-helix AH2 and begins a sequence of four highly conserved residues, GEHT, which are shown in a box in Figure 4. The outer brackets show the region of the protein previously defined as the B domain (Giuliani et al. Infect Immun 2005 ;73: 1151- 60).
[00226] A second chimeric fHBP, referred to herein as Chimera II, was generated.
Chimera II was identical in amino acid sequence to Chimera I except for the introduction the A174K substitution (Bold and underlined K, Chimera II, Figure 4). It should be noted that the A domain is not shown in Figure 4, or in the NMR-based structure of Figure 5, but would be attached at the N-terminus labeled "N" in Figure 5.
[00227] Models of the two chimera vaccines are shown in Figure 5, Panels A and B.
Chimera I contains the JAR 11 epitope, including residue A174 (Figure 5, Panel A). Chimera II contains the JAR 32 and JAR 35 epitopes, including residue K174 (Figure 5, Panel B). The model of the fHBP chimeras was constructed based on the NMR structure of Cantini et al. J Biol Chem 2006;281:7220-7.
EXAMPLE 4: PURIFICATION AND CHARACTERIZATION OF MUTANT PROTEINS.
[00228] Recombinant proteins were expressed in E. coli as C-terminal hexahistidine
(HiS6) fusions, which were purified by metal chelate chromatography. Specifically, Proteins were expressed from pET21-based plasmids in E. coli BL21(DE3) as C-terminal hexahistidine fusions. Fusion proteins were then isolated by metal chelate chromatography according to methods known in the art. Isolated proteins were dialyzed against Ix PBS, 5% sucrose, 1 mM DTT and filter sterilized. Proteins (5 μg each) were separated on a 4-12% polyacrylamide gel and stained with Coomassie blue.
[00229] The results are shown in Figure 6. Lane 1, mass standard; 2, fHBP v.l
(MC58); 3, fHBP v.2 (8047); 4, fHBP Chimera I; 5, fHBP Chimera II.
EXAMPLE 5: EPITOPE EXPRESSION BY CHIMERIC ANTIGENS.
[00230] ELISA was used to assess concentration-dependent binding of the anti-fHBP mAbs to the chimeric antigens isolated in Example 4. As expected, JAR 1, which binds to a v.l epitope in the C domain (R204), did not bind to either of the chimeric proteins (Fig. 7, Panel A). JAR 5, which is specific for an epitope on the B domain of fHBP v.l, and JAR 4, which cross-reacts with an epitope that is not yet defined by expressed by v.l and v.2, showed identical respective concentration-dependent binding with the two chimeric proteins as compared with the respective wild-type v.l and/or v.2 proteins.
[00231] Figure 7, Panel B, provides binding data for mAbs JAR 10, 11 and 13, which were from a mouse immunized with v.2 or fHBP. All three mAbs recognize epitopes on the C domain of fHBP v.2 of strain 8047 (Figure 20), and they showed similar respective concentration-dependent binding with the Chimera I protein as they did with the wild-type rfHBP v.2 control protein expressed from the gene of strain 8047 (Fig. 7, Panel B). As expected, JAR 11 did not bind to Chimera II, since this protein had lysine substituted for alanine at position 174 (A 174K), which eliminated the JAR 11 epitope and introduced the JAR 32 epitope (Panel C). JAR 36, which cross-reacts with an epitope not yet defined but present on fHBP v.2 and v.3 fHBP bound to both of the chimeric proteins, and to the wild- type rfHBP v.2 control but not with the fHBP v.l control (Fig. 7, Panel C). Collectively, the data showed that the two chimeric fHBPs expressed epitopes associated with fHBP v.l, v.2, and/or v.3 proteins, and reacted as expected with the various mAbs in accordance with our studies localizing the epitopes.
EXAMPLE 6: IMMUNIZATION OF MICE WITH DOUBLE MUTANT AND CHIMERIC FHBPS AND BACTERICIDAL ANTIBODY RESPONSES
[00232] The proteins shown in Figure 6 were used to immunize mice according to the schedule in Table 1, below. Four doses of vaccine (20 μg of protein) were administered IP with intervals of 2 weeks between doses. Mice were bled 2.5 weeks after dose 4. CFA = complete Freund's adjuvant; IFA = incomplete Freund's adjuvant; Al(OH)3 = aluminum hydroxide. CFA/IFA below indicates that the mice received an initial dose with CFA, then subsequent booster doses with IFA. Where Al(OH)3 was used as the adjuvant, all doses were administered with aluminum hydroxide as the adjuvant.
Table 1. Immunization schedule1
Group fHBP Protein Adjuvant
1 CFA/IFA
2 fHBP v.l CFA/IFA 3 fHBP v.2 CFA/IFA 4 Chimera I CFA/IFA 5 Chimera II CFA/IFA
6 Al(OH)3 7 fHBP v.l Al(OH)3 8 fHBP v.2 Al(OH)3 9 Chimera I Al(OH)3 10 Chimera II Al(OH)3
Groups l to 10 consist of 5 CD-I mice each (N=60). Each animal received four injections
[00233] Figure 9 summarizes the serum antibody responses of the mice that received the chimeric vaccines administered with FA when measured against strain H44/76 (variant 1) and four additional strains that express subvariants of v.l fHBP (i.e., fHBP proteins with relatively small amino acid differences (e.g., with greater than about 88%, and less than 97% amino acid sequence identity) between the sequence of the respective protein and that of v.1.1 fHBP of H44/76). The mice immunized with the wild-type rfHBP v.l control protein had high responses to H44/76 (reciprocal GMT of nearly 10,000) and lower and variable responses against the other four test strains (ranging from a GMT of <l:10 against strain NZ98/254 to a GMT of >l:l,000 against SK084). The sera from the mice immunized with the rfHBP v.2 control protein were either negative for bactericidal activity (bactericidal titers <l:10, four strains) or weakly positive (reciprocal GMT of 1:10; strain SK141). The mice immunized with either chimeric protein vaccine developed serum bactericidal antibodies against all four strains that were susceptible to bactericidal activity of sera from the control mice immunized with rfHBP v.1. For three of the four strains, the respective reciprocal GMTs of the chimeric vaccine groups were ~ 1 log lower than those of mice given the control rfHBP v.l vaccine whereas the responses to the fifth strain, SK141, were as high or higher than those of the mice given the control rfHBP v.l vaccine.
[00234] Figure 10 summarizes the serum bactericidal antibody responses as measured against the six strains expressing fHBPs in the v.2 or v.3 variant groups. Three of the strains were JAR 11 positive (left panel) and three were JAR 32 positive (right panel). Sera from control mice immunized with rfHBP v.2 were bactericidal against all six strains whereas, with one exception (strain 03S-0658), the serum bactericidal titers of control mice immunized with rfHBP v.l were <l:10. The sera from the mice immunized with either chimeric vaccine were bactericidal against all six strains. For four of the strains (8047, MD1321, M1239 and SK104), the respective titers were ~1 log lower than those of the control mice immunized with rfHBP v.2. For the remaining two strains, 03S-0658 and MD1435) the titers elicited by one or both chimeric vaccines were similar to those of the mice given the positive control rfHBP v.2 vaccine. Thus, in contrast to the control rfHBP v.l or v.2 vaccines, the chimeric vaccines elicited bactericidal antibody responses against strains expressing fHBP from each of the three antigenic variant groups.
[00235] The Chimera I vaccine expressed the JAR 11 epitope while the Chimera II vaccine expressed the JAR 32 epitope (Fig. 1). However, with one exception, the respective responses of mice immunized with either chimeric vaccine when measured against strains expressing JAR 11- or JAR 32-positive fHBP were not significantly different from each other (Fig. 5). The exception was the JAR 11-positive strain MD1435, where there was a trend for a higher reciprocal GMT in the group of mice immunized with Chimera I than Chimera II (P =0.06).
[00236] Thus, the A174K substitution in Chimera II that eliminated the JAR 11 epitope (v.2) and introduced the JAR 32/35 epitope (v.3) did not appreciably increase the bactericidal responses against a test strain expressing the v.3 protein. [00237] Figure 11 summarizes the serum bactericidal antibody responses of mice immunized with the chimeric vaccines when absorbed with aluminum hydroxide as measured against the panel of test strains with v.l fHBP. The corresponding data for responses to test strains expressing v.2 or v.3 fHBP are shown in Figure 12. In general the respective responses to the different vaccines absorbed with aluminum hydroxide paralleled those observed to the vaccines given with Freund's adjuvant although as expected the titers were somewhat lower with the aluminum hydroxide adjuvant.
[00238] The data above indicate that the JAR3/5 epitope, which is common to virtually all v.l proteins but is not present in fHBP from v.2 or v.3 strains, is important for eliciting high bactericidal antibody titers to the variant 1 fHBP protein. This discovery that the epitope recognized by JAR 3/5 plays an important role in eliciting v.1 fHBP bactericidal antibodies provides the basis for the rational design of further chimeric fHBP vaccines that can elicit bactericidal antibodies across strains expressing different fHBP protein variants, particularly against both v.l and v.2 fHBP-expressing strains. For example, the two chimeric vaccines described above (Chimera I and Chimera II) included, from N-terminus to C-terminus:
1) an A domain (common to both v.l and v.2);
2) a heterologous B domain composed of, from N-terminus to C-terminus, a) a contiguous amino acid sequence of an N-terminal portion of a B domain of a v.l fHBP containing the amino acid sequence defining the JAR 3/5 epitope, operably linked to b) a contiguous amino acid sequence defining the remainder of the B domain, which amino acid sequence is that of a B domain of a v.2 fHBP, where the v.2 fHBP amino acid sequence is present in the heterologous B domain following the amino acid sequence of GEHT; and
3) a C domain of the v.2 protein. EXAMPLE 8: NATURAL OR SYNTHETIC POLYMORPHISMS OF v.l FHBP B DOMAINS
[00239] As noted above, the discovery that the JAR 3/5 epitope in the N-terminal portion of v.1 fHBP provides the basis for construction of additional chimeric fHBP polypeptides that can serve as vaccines to elicit cross-reactive, bactericidal antibodies against v.l fHBP-expressing N. meningitidis strains and v.2 fHBP-expressing N. meningitidis strains. In order to provide guidance as to the amino acid sequences of v.l fHBP that find use in the fHBP chimeric vaccines contemplated here, various v.l fHBP amino acid sequences of the B domain were analyzed.
[00240] Figure 13 provides an alignment of fHBP v.l sequences with natural polymorphisms in the Ν-terminal portion of the B domain. The sequence conservation is shown below the alignment, with "*" denoting residues that are identical, ":"denoting residues that are conserved, and "."denoting residues that are semi-conserved across the aligned sequences. The positions of alpha-helices are shown above the alignment, with the residues implicated in the alpha helices indicated by italics. The residue, G121, which is implicated in the JAR 3 and JAR 5 epitope, is shown in bold and underlining. A lysine at position 122 (K122) also appears to be important in the JAR 3/5 epitope, since one strain that is negative for JAR 3 and JAR 5 binding, 03S-0408, has G121 but differs from JAR 3/5 reactive strains in having serine at position 122 (S122). (data not shown) The amino acid sequence of GEHT that provides the point at which the B domain sequence of the chimera "switches" or "crosses over" to the amino acid sequence of a v.2 B domain in Chimera I and Chimera II (referred to herein as the "junction point") is shown in the box. [00241] As shown in Figure 13, there are a number of natural polymorphisms in the region of G121, which is involved in the JAR 3/JAR 5 epitope. Notably, a fHBP containing R121 (rather than G121) does not express the JAR 3/JAR 5 epitope. However, polymorphisms near G121, for example Pl 17 in NZ98/254 and D121 in 4243, do not interfere with the binding of JAR 3 or JAR 5. Thus, some modifications at residues other than G121 provide for JAR 3/5 epitope expression.
[00242] The alignment thus provides guidance as to the types of amino acid substitutions that can be introduced while maintaining JAR 3/5 epitope expression. Such amino acid substitutions can be either naturally-occurring (i.e., natural polymorphisms) or can be introduced by synthetic means (e.g., recombinant polymorphisms). Thus, the heterologous B domains of the chimeric fHBP polypeptides contemplated herein include those containing v.1 B domain sequences with natural and synthetic polymorphisms in the proximal B domain, including both natural and synthetic variants.
EXAMPLE 9: NATURAL OR SYNTHETIC POLYMORPHISMS OF FHBP C DOMAINS
[00243] In addition, to the JAR 3/5 epitope, chimeric fHBPs contemplated herein generally include one or more epitopes of v.2 fHBP B and C domains, where the v.2 fHBP B domain epitopes are present at a location C-terminal to (i.e., distal to) the location of the JAR 3/5 epitope of v.l fHBPs, which is generally C-terminal to the alpha helix that follows the sequence of GEHT, which is shared between v.l and v.2 fHBPs. In order to provide guidance as to the amino acid sequences of v.2 fHBP that find use in the fHBP chimeric vaccines contemplated here, various v.2 fHBP amino acid sequences of the B domain, as well as the C domain were analyzed.
[00244] Figure 14 provides an alignment of exemplary v.2 fHBP sequences of the distal portion of the B domain, and further provides exemplary v.2 fHBP sequences of the C domain, where the B domain is generally defined as residues 101 to 164, with numbering based on MC58 fHBP as a reference.
[00245] Therefore, based on the polymorphic variants shown in Figures 13 and 14, variations in the amino acid sequence of v.2 fHBP B domains and C domains, as well as additional combinations of v.l/v.2 heterologous B domains with different v.2 C domains to can be readily envisioned to generate additional chimeric fHBPs. Figures 13 and 14 provide 7 exemplary amino acid sequences of the N-terminal region of the v.l B domain (Figure 10) and 9 exemplary amino acid sequences for the distal portion of the B domain of v.2 fHBP and for the C domain of v.2 fHBP (Figure 11). Thus, the sequences in Figures 13 and 14 provide for a least 63 different fHBP chimeras, simply by combining the amino acid sequences as provided. Further, the alignments provide guidance as to the amino acid residues that can be substituted.
[00246] Thus, not only do these exemplary sequences provide guidance for use of amino acid sequences containing naturally-occurring polymorphisms, they also provide guidance for production of synthetically-generated variants (e.g., using recombinant methods). For example, additional point mutations may be introduced into the coding nucleic acid to provide for fHBP chimeras that express other desirable epitopes, such as the v.l epitope recognized by JAR 1/mAb 502 that is in the portion of the C domain (R204), or the JAR 11 epitope in Chimeras IV and V. Introduction of the JARl/mAb 502 epitope can provide for improved cross-reactivity against v.l strains from the ST-32 lineage that nearly always express the JAR 1 epitope, while eliciting antibodies to the JAR 11 epitope can provide for improved titers against v.2 strains (see Koeberling et al., J Infect Dis 2008, 198:262-270, for a description of v.l.1).
[00247] Note also, that despite engineering expression of the JAR 11 epitope in
Chimera I, and the JAR 32 epitope in Chimera II, no statistically significant differences were observed in the respective serum bactericidal antibody responses of mice immunized either vaccine against strains expressing v.2 or v.3 fHBP that were JAR 11-positive or JAR 32- positive. However, the discovery that binding of antibody to an epitope located near residue 174 (i.e., JAR 11 in some strains, and JAR 32 in others; see Figure 3 ) was not sufficient to elicit complement-mediated bactericidal activity in the absence of eliciting additional antibodies that bind to a second epitope associated with ion pair at residues 180 and 192 (such as JAR 10 in some strains or JAR 33 in others (Table in Figure 20 ) indicates that coverage can be improved against JAR 32-positive strains by engineering expression of a second epitope defined by binding with JAR 33 (i.e., R180/E192). Note that among wild-type strains expressing fHBP v.2 or v.3, expression of JAR 32 is often associated with expression of JAR 33, while expression of JAR 11 is usually associated with expression of JAR 10 (see, for example, strain panel, Figure 8).
EXAMPLE 10: FURTHER EXEMPLARY CHIMERIC VACCINES.
[00248] Table 2 and Figures 15 and 16 provide for additional hypothetical chimeric vaccines, designated Chimera III, Chimera IV and Chimera V. Each of these chimeric fHBPs are generated using a strategy similar to that used to generate Chimera I to provide for a chimeric fHBP that elicits bactericidal anti-v.l and v.2 antibodies.
[00249] As illustrated in Figure 15, Chimera III contains the A domain and proximal B domain of subvariant v.l.10 encoded by the fHBP from NZ98/254, and uses the same distal
B domain and C domain of Chimera I and Chimera II.
[00250] Chimera IV, illustrated in Figure 15, includes the A and proximal B domains of Chimera I and Chimera II, but substitutes the distal B and C domains of v.2 from strain
8047 (v.2.1) of these chimera with those from RM1090 (v.2 subvariant).
[00251] Chimera V fuses the A and proximal B domains of a v.1 subvariant (strain
NZ98/254) with that distal B and C domains of v.2 subvariant (strain RM1090). [00252] The amino acid changes that will result in the respective Chimera III, IV and
V vaccine antigens, as compared with the respective amino acid sequences of the A and proximal B domain of MC58 and distal B and C domains of 8047 used to prepare Chimera 1 are shown in Figure 16. Chimeras III and V can provide for increased breadth of protection against certain strains expressing subvariants of v.l, such as NZ98/254, which is not covered by the Chimera I or II vaccines. Chimeras IV and V can provide for extended protection against v.3 strains, which are poorly covered by Chimera I or Chimera II. [00253] Table 2 provides a summary of the observed epitope expression for Chimera I and Chimera II, and further sets out the predicted epitope expression for proposed Chimeras III, IV, and V.
Figure imgf000070_0001
[00254] Further modifications of the chimeric fHBP include varying the residue within the heterologous B domain at which the v.l fHBP B domain sequence ends and the v.2 (or v.2) fHBP B domain sequence ends. Specifically, although residue G136 was used as the position of the "crossover" between fHBP variants, the "crossover" residue can be any residue C-terminal of the amino acid sequence defining the JAR 3/JAR 5 epitope and a residue within M123-S136 can also be selected. In addition, residue positions C-terminal to G136 (E137-D142), or residue positions adjacent or within α-helix AH2 (K143-A150, preceding the first beta-strand) can be suitable crossover residue positions. Therefore, a number of different crossover residue positions are contemplated herein, where the crossover residue may be positioned after G121 of the JAR 3/5 epitope (G121) through and including A 174 of the JAR 11 epitope, where the presence of the JAR 3/5 epitope can be assessed using immunoassay methods known in the art, (Note that numbering above is based on the amino acid sequence number of the fHBP reference sequence of MC58.)
EXAMPLE 11: EXPRESSION OF CHIMERIC FHBP IN N. MENINGITIDIS
[00255] Figure 33 shows a Western blot of samples of N. meningitidis expressing either wildtype (WT) or a chimeric fHBP (Chimera I (see Fig. 23)). The Chimera I corresponded to a full-length fHBP and further included the signal sequence (but lacked any epitope tags, such as a Penta-His tag) As shown in Figure 33, bacterial cells transformed with the plasmid containing the gene encoding Chimera I were positive for both anti-fHbp mAbs, whereas the cells from H44/76 transformed with the plasmid containing the gene encoding fHbp v.2 or WT H44/76 react only with JAR 3 (v.l) and the cells from 8047 react only with JAR 13 (v.2).
ATCC DEPOSIT
[00256] Hybridomas producing the JAR 4, JAR 5, JAR 11, and JAR 32 monoclonal antibodies were deposited under the terms of the Budapest Treaty with the American Type Culture Collection, 10801 University Blvd., Manassas, Va. 20110-2209, USA (ATCC) on the date indicated in the table below, and were assigned the designations set out in the table below.
Figure imgf000071_0001
[00257] It should be noted that JAR 5 mAb specifically binds to an epitope that at least overlaps with the epitope specifically bound by JAR 3 mAb, and that JAR 32 mAb specifically binds to an epitope that at least overlaps with the epitope specifically bounds by JAR 35 mAb.
[00258] These deposits were made under the provisions of the Budapest Treaty on the
International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations there under (Budapest Treaty). This assures maintenance of a viable culture of the deposit for 30 years from the date of deposit and for at least five (5) years after the most recent request for the furnishing of a sample of the deposit received by the depository. The deposits will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Children's Hospital & Research Center at Oakland and the ATCC (the assignee of the present application) which assures that all restrictions imposed by the depositor on the availability to the public of the deposited material will be irrevocably removed upon the granting of the pertinent U.S. patent, assures permanent and unrestricted availability of the progeny of the culture of the deposit to the public upon issuance of the pertinent U.S. patent or upon laying open to the public of any U.S. or foreign patent application, whichever comes first, and assures availability of the progeny to one determined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto according to 35 U.S.C. § 122 and the Commissioner's rules pursuant thereto (including 37 C.F.R. § 1.14 with particular reference to 886 OG 638). [00259] The assignee(s) of the present application has agreed that if a culture of the materials on deposit should die or be lost or destroyed when cultivated under suitable conditions, the materials will be promptly replaced on notification with another of the same. Availability of the deposited material is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.

Claims

CLAIMS WHAT IS CLAIMED IS:
1. A chimeric factor H binding protein (fHBP) comprising, from N-terminus to C- terminus: a) a heterologous B domain comprising, from N-terminus to C-terminus, i) a contiguous N-terminal amino acid sequence of a variant 1 (v.l) factor
H Binding Protein (fHBP) B domain; and ii) a contiguous C-terminal amino acid sequence of a variant 2 (v.2) fHBP
B domain or a variant 3 (v.3) fHBP B domain; and b) a C domain operably linked to the heterologous B domain of a), wherein the C domain comprises a contiguous amino acid sequence of a variant 2 (v.2) fHBP C domain or a variant 3 (v.3) fHBP C domain; wherein the chimeric fHBP specifically binds a JAR 5 monoclonal antibody and specifically binds a monoclonal antibody specific for an epitope of a v.2 or v.3 fHBP C domain.
2. The chimeric fHBP of claim 1, wherein the contiguous N-terminal amino acid sequence of the v.1 fHBP B domain comprises an amino acid sequence at least 80% identical to the contiguous amino acid sequence of
QSHSALTAFQ TEQIQDSEHS GK (SEQ ID NO: 1), LTAFQ TEQIQDSEHS GKMVAKRQFR IGDIA, TEQIQDSEHS GKMVAKRQFR IGDIAGEHTA FNQLPD, or QSHSALTAFQ TEQIQDSEHS GKMVAKRQFR IGDIAGEHTA FNQLPD.
3. The chimeric fHBP of claim 2, wherein the amino acid sequence of the heterologous B domain comprises one or more of: a substitution of leucine (L) for the phenylalanine (F) at residue position 9 of QSHSALTAFQ TEQIQDSEHS GK (SEQ ID NO: 1); a substitution of a valine (V) or glutamic acid (E) for isoleucine (I) at residue position 4 of SEQ ID NO: 1; a substitution of a proline (P) for serine (S) at residue position 17 of SEQ ID NO: 1; a substitution of an aspartic acid (D) for histidine (H) at residue position 19 of SEQ ID NO:1; a substitution of an arginine (R) for glutamine (Q) at residue position 28 of SEQ ID NO:1; a substitution of valine (V) for alanine (A) at residue position 35 of SEQ ID NO:1; a substitution of glycine (G) for aspartic acid (D) at residue position 42 of SEQ ID NO: 1; and a substitution of lysine (K) for glutamic acid (E) at residue position 46 of SEQ ID NO:1.
4. The chimeric fHBP of claim 1, wherein the chimeric fHBP comprises an amino acid sequence of an A domain of a v.l, v.2 or v.3 fHBP positioned N-terminal of and operably linked to the heterologous B domain.
5. The chimeric fHBP of claim 1, wherein the chimeric fHBP comprises at least one epitope that when bound by an antibody blocks binding of fH to fHBP.
6. The chimeric fHBP of claim 1, wherein the chimeric fHBP comprises a pair of epitopes that elicit antibodies that, when bound to their respective epitopes, exhibit bactericidal activity.
7. The chimeric fHBP of claim 6, wherein the pair of epitopes is a JAR 10 monoclonal antibody epitope and a JAR 11 monoclonal antibody epitope.
8. The chimeric fHBP of claim 6, wherein the pair of epitopes is a JAR 33 monoclonal antibody epitope and a JAR 35 monoclonal antibody epitope.
9. A chimeric factor H binding protein (fHBP) comprising, from N-terminus to C- terminus: a) a first amino acid sequence at least 80% identical to a contiguous amino acid sequence of a variant 1 (v.l) factor H binding protein (fHBP) B domain comprising the amino acid sequence QSHSALTAFQ TEQIQDSEHS GK (SEQ ID NO:1); and b) operably linked to a), a second amino acid sequence at least 80% identical to a contiguous amino acid sequence of a variant 2 (v.2) or variant 3 (v.3) fHBP; wherein the first and second amino acid sequences are operably linked at a junction point positioned within a B domain or a C domain of the chimeric fHBP; wherein the chimeric specifically binds a monoclonal antibody specific for an epitope of a v.2 or v.3 fHBP C domain.
10. The chimeric fHBP of claim 9, wherein the junction point is positioned within the B domain such that the chimeric fHBP comprises a heterologous B domain.
11. The chimeric fHBP of claim 9, wherein the junction point is positioned within the C domain such that the chimeric fHBP comprises a heterologous C domain.
12. The chimeric fHBP of claim 9, wherein the second amino acid sequence is greater than 80% identical to a contiguous amino acid sequence of a v.2 fHBP and the junction point is positioned at a site following the lysine of SEQ ID NO: 1 of the first amino acid sequence and prior to a residue defining a JAR 11 monoclonal antibody epitope.
13. The chimeric fHBP of claim 9, wherein the second amino acid sequence is greater than 80% identical to a contiguous amino acid sequence of a v.3 fHBP and the junction point is positioned at a site following the lysine of SEQ ID NO: 1 of the first amino acid sequence and prior to a residue defining a JAR 32 monoclonal antibody epitope.
14. The chimeric fHBP of claim 9, wherein the junction point is positioned within the B domain adjacent or within the second alpha helix.
15. The chimeric fHBP of claim 14, wherein the junction point is positioned C- terminal of the amino acid sequence GEHT of the B domain.
16. The chimeric fHBP of claim 9, wherein the junction point is positioned at a site that is not more than 14 residues N-terminal of the amino acid sequence GEHT.
17. The chimeric fHBP of claim 9, wherein the junction point is positioned at site not more than 34 residues C-terminal of the amino acid sequence GEHT.
18. The chimeric fHBP of claim 9, wherein the junction point is positioned so as to provide a heterologous B domain comprising an amino acid sequence at least 80% identical to the amino acid sequence
TEQIQDSEHS GKMVAKRQFR IGDIAGEHTA FNQLPD.
19. The chimeric fHBP of claim 9, wherein the chimeric fHBP comprises a heterologous B domain comprising an amino acid sequence at least 80% identical to the amino acid sequence
QSHSALTAFQ TEQIQDSEHS GKMVAKRQFR IGDIAGEHTA FNQLPD.
20. The chimeric fHBP of claim 9, wherein the junction point is positioned such that the chimeric fHBP comprises a heterologous C domain, wherein the heterologous C domain comprises an N-terminal amino acid sequence of KLTYTIDFA.
21. The chimeric fHBP of claim 9, wherein the chimeric fHBP comprises at least one epitope that elicits an antibody that blocks binding of fH to fHBP.
22. The chimeric fHBP of claim 9, wherein the chimeric fHBP comprises a pair of epitopes that elicit antibody pairs that, when bound to their respective epitopes, exhibit bactericidal activity.
23. The chimeric fHBP of claim 22, wherein the pair of epitopes is a JAR 10 monoclonal antibody epitope and a JAR 11 monoclonal antibody epitope.
24. The chimeric fHBP of claim 22, wherein the pair of epitopes is a JAR 33 monoclonal antibody epitope and a JAR 35 monoclonal antibody epitope.
25. The chimeric fHBP of claim 9, wherein the chimeric fHBP comprises an amino acid sequence of an A domain of a v.l, v.2 or v.3 fHBP positioned N-terminal of and operably linked to the heterologous B domain.
26. A nucleic acid encoding the chimeric fHBP of claim 1.
27. A nucleic acid encoding the chimeric fHBP of claim 9.
28. A recombinant host cell containing the nucleic acid of claim 26.
29. A recombinant host cell containing the nucleic acid of claim 27.
30. A method of producing a chimeric fHBP, the method comprising: culturing the recombinant host cell of claim 28 under conditions suitable for expression of the chimeric fHBP; and isolating the chimeric fHBP.
31. A method of producing a chimeric fHBP, the method comprising: culturing the recombinant host cell of claim 29 under conditions suitable for expression of the chimeric fHBP; and isolating the chimeric fHBP.
32. An immunogenic composition comprising a chimeric fHBP according to claim 1, and a pharmaceutically acceptable excipient.
33. The immunogenic composition of claim 32, wherein the chimeric fHBP is in a vesicle preparation prepared from a Neisseria meningitidis strain.
34. An immunogenic composition comprising a chimeric fHBP according to claim 9, and a pharmaceutically acceptable excipient.
35. The immunogenic composition of claim 34, wherein the chimeric fHBP is in a vesicle preparation prepared from a Neisseria meningitidis strain.
36. A method of eliciting an antibody response in a mammal, the method comprising administering to a mammal a chimeric fHBP according to claim 1.
37. The method of claim 36, wherein the mammal is a human.
38. The method of claim 37, wherein said administering provides for production of antibodies that bind a factor H binding protein (fHBP) of a variant 1 N. meningitidis strain and that bind a fHBP of a variant 2 N. meningitidis strain.
39. A method of eliciting an antibody response in a mammal, the method comprising administering to a mammal a chimeric fHBP according to claim 9.
40. The method of claim 39, wherein the mammal is a human.
41. The method of claim 39, wherein said administering provides for production of antibodies that bind a factor H binding protein (fHBP) of a variant 1 N. meningitidis strain and that bind a fHBP of a variant 2 N. meningitidis strain.
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US20120107339A1 (en) 2012-05-03
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