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

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

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority benefit of U.S. provisional application Ser. No. 61/035,329, filed Mar. 10, 2008 and U.S. provisional application Ser. No. 61/037,252, filed Mar. 17, 2008, both of which are incorporated herein by reference in their entirety.

This invention was made with government support under Public Health Service grant nos. R01 AI46464 and C06 RR16226. The government has certain rights in this invention.

FIELD OF THE INVENTION

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.

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.1), 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.1). 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.1 and v.2) the conservation can be as low as 59%. The protein is expressed by all known strains of N. meningitidis.

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.1 confers protection against N. meningitidis strains expressing fHBP v.1, 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.1 (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.

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 GNA2091 protein and a GNA1870 (or “fHBP”) protein, and a second fusion protein of a GNA2132 protein and a GNA1030 protein (Giuliani et al. Proc Natl Acad Sci USA 2006, 103:10834-9). A third strategy has been to construct a fusion of different serologic variants (“serovars”) of one antigen to induce cross-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).

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.

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.).

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.1 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.

SUMMARY

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

FIG. 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 -A1). 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.

FIG. 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.1 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 FIG. 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 FIG. 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.1 fHBP lacking the signal sequence (Masignani et al., 2003 J Exp Med 197:789-99).

FIG. 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.1 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.1 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.

FIG. 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.1 and the JAR 10 epitope, which is on the C domain of subsets of strains expressing v.1, 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.

FIG. 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 1× 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.1 (MC58); 3, fHBP v.2 (8047); 4, fHBP Chimera I; 5, fHBP Chimera II.

FIG. 7 is a set of graphs illustrating binding of individual anti-fHBP mAbs to recombinant proteins. Panel A shows mAbs prepared against fHBP v.1; Panel B, fHBP v.2; Panel C, fHBP v.3. The symbols represent different antigens on the plate: open squares, fHBP v.1; open circles, fHBP v.2; open triangles, Chimera I; asterisks, Chimera II.

FIG. 8A 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.1, v.2 and v.3 and JAR mAb binding of the respective fHBPs.

FIG. 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.

FIG. 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.1 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.1 (See Table in FIG. 8A, above). Values are presented as 1/GMT (Reciprocal (or inverse) geometric mean titer) with a 95% confidence interval.

FIG. 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 FIG. 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.

FIG. 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.1 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.1. 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 FIG. 10.

FIG. 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 FIG. 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 FIG. 10.

FIG. 13 is a schematic showing alignment of fHBP v.1 amino acid sequences with natural polymorphisms in the N-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 fHbpN domain and a small N-terminal portion of the fHbpC domain as indicated above the alignment. The sequence conservation is shown below the alignment (code as in FIG. 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 5122. 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 JARS/5 reactivity.

FIG. 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 FIG. 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.

FIG. 15 is a schematic illustrating additional exemplary chimeric vaccines (Chimeras IIb, III, IV, and V). Chimera IIb can be made by introducing the K180R 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 RM1090.

FIG. 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.

FIG. 17 provides a table summarizing cross-reactivity of the different JAR mAbs, their respective Ig isotypes and ability to inhibit binding of human fH.

FIG. 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.

FIG. 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.

FIG. 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.

FIG. 21 provides the amino acid sequence of variant 1 (v.1) 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.

FIG. 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.

FIG. 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).

FIG. 24 is a schematic showing Chimera II (MC58/8047 A174K) 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.

FIG. 25 is a schematic showing Chimera IIb (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 K180R substitutions are shown in bold text.

FIG. 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).

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).

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).

FIG. 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).

FIG. 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).

FIG. 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).

FIG. 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.

FIG. 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.1) WT cells; 6, 8047 (v.2) WT cells; 7, 8047 KO fHBP cells; 8, recombinant (r) fHBP v.1 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.1); lower panel, blot probed with anti-fHBP mAb JAR 13 (v.2 or v.3).

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 N-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 N-terminal domain containing a mix of α helices and β strands is named the fHbpN domain (left) and the C-terminal domain consisting of β strands is labeled as the fHbpC domain (right). The fHbpN 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 NMR structure of Cantini et al. J Biol Chem 2009.

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

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.

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.

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.

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

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.

“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.1), 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.1, 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 FIG. 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 FIG. 4 for alignment.

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.1 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.1 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 Neisserial amino acid or nucleic acid sequence of one strain is heterologous to a Neisserial host of another strain.

“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.

“Derived from” in the context of an amino acid sequence or polynucleotide sequence (e.g., an amino acid sequence “derived from” a v.1 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.

“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, gln; 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.

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).

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.

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.

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.

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.

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 “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

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.1 fHBP of the N. meningitidis strain MC58 was arbitrarily selected as a reference sequence for all native v.1, 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 fHbpN and the fHbpC. Details of each of these domains with reference to the amino acid sequence of v.1 fHBP of MC58 strain is described below.

A, B, and C Domains Using First Definition.

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.1 fHBP of the MC58 strain along with the boundaries of the A, B and C domains is shown in FIG. 21. The Q101 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 AH1 and AH2). Residues GEHT are underlined followed by the second a 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 N-terminal portion of the B domain are not shown). The native v.1 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 a 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

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.1 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 a-helix AH2 as “α” below the sequence in FIG. 21 marks the positions of a 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.

fHBP has been divided into three variant groups (referred to as variant 1 (v.1), 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.1) (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.1; 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.1, 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 FIGS. 8A and 8B.

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, J. 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 DNA 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. 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 DNA sequences of seven housekeeping genes in Neisseria meningitidis strains (Maiden et al., 1998, Proc. Natl. Acad. Sci. USA 95:3140).

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.

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.

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 N98/254); 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).

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.

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.

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.

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. Mol. 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.

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.

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 50° C. or higher and 0.1×SSC (15 mM sodium chloride/1.5 mM sodium citrate). Another example of stringent hybridization conditions is overnight incubation at 42° C. in a solution: 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt\'s solution, 10% dextran sulfate, and 20 mg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC at about 65° C. 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.

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 of fHBPs

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 Q101 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.1 fHBP of MC58, where the numbering is based on amino acid sequence of MC58 v.1 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.1 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.1 fHBP).

B Domain of v.1 fHBP

As noted above, the chimeric fHBPs of the present disclosure contain amino acid sequence of a v.1 fHBP B domain. Amino acid sequences of v.1 fHBP, including v.1 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.1 fHBPs. FIG. 8A illustrates the amino acid sequence identity between full length fHBPs of exemplary v.1, v.2 and v.3 strains, and further illustrates the presence or absence of epitopes defined by the indicated JAR mAbs. FIG. 8B illustrates the amino acid sequence identity between the A, B, and C domains of exemplary v.1, 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.

FIG. 21 shows the amino acid sequence of the fHBP of the v.1 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 a -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.1 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.1 fHBP lacking the signal sequence (Masignani et al., 2003 J Exp Med 197:789-99).

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.

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.1 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.1 fHBP.

B and C Domains of v.2 fHBP and of v.3 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. For convenience and clarity, the C domain can be structurally defined as those residues corresponding to residues 165-255 of v.1 fHBP of MC58, where the numbering is based on amino acid sequence of MC58 v.1 fHBP lacking the signal sequence (Masignani et al., 2003 J Exp Med 197:789-99) (see FIG. 21).

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.

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.1 fHBP generally is about 64 residues in length.

fHbpN Domain of fHBPs

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.1 fHBP of MC58, where the numbering is based on amino acid sequence of MC58 v.1 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.1 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.

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.

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.1 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.1 fHBP.

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.1 fHBP).

As noted above, the chimeric fHBPs of the present disclosure contain amino acid sequence of a v.1 fHbpN domain. Amino acid sequences of v.1 fHBP, including v.1 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.1 fHBPs. The alignment illustrates the position and identity of naturally occurring polymorphisms among v.1 fHBPs. FIG. 8A illustrates the amino acid sequence identity between full length fHBPs of exemplary v.1, v.2 and v.3 strains, and further illustrates the presence or absence of epitopes defined by the indicated JAR mAbs. FIG. 8B illustrates the amino acid sequence identity between exemplary v.1, 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

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 a-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.3 fHBP

Exemplary chimeric fHBPs of the present disclosure contain heterologous domains comprising a full or partial fHbpN domain of a v.1 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.1 fHBP of MC58, where the numbering is based on amino acid sequence of MC58 v.1 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.

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.1 fHBP.

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.

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.

The term “chimeric factor H binding protein” or “chimeric fHBP” refers to a polypeptide comprising, from N-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 N-terminal portion derived from a contiguous amino acid sequence of a v.1 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 N-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.

Where the chimeric fHBP contains a heterologous B domain, the heterologous B domain generally comprises at least an N-terminal portion derived from a contiguous amino acid sequence of a v.1 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 N-terminal portion for which a corresponding contiguous amino acid sequence of a v.1 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.

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 N-terminal amino acid sequence of a B domain of a v.1 fHBP.

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.1 fHBP B domain operably linked to heterologous C domain comprising at least an N-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.1 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. 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.

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.

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).

Exemplary chimeric fHBPs are described in detail below.

Exemplary Chimeric fHBPs

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

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.1 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.

In general, a B domain amino acid sequence comprising an amino acid sequence N-terminal to the second a 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.1 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.

Where the chimeric fHBP has a heterologous B domain, the junction point may be positioned at a residue adjacent to the second a helix (AH2) (e.g., adjacent and C-terminal to a residue corresponding to residue 121 or 122 of FIG. 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.

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.1 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.

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.1 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.

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.1 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.1 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).

For example, where the heterologous B domain includes residues 101-122 or a v.1 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.1 sequences exemplified above.

In another embodiment, the junction point is positioned N-terminal to the second a helix (AH2), which are denoted in FIG. 22 by “a”. As pointed out above, the residues GEHT are highly conserved across v.1, 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.

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.1 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.

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

(SEQ ID NO: 1) QSHSALTAFQ TEQIQDSEHS GK 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 (G/E/R)K.

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.

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)

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 seuence of

TEQIQDSEHS GKMVAKRQFR IGDIAGEHTA FNQLPD, where the amino acid sequence optionally provides for an epitope that mediates specific binding of a JAR 5 mAb.

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

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