CROSS-REFERENCE TO RELATED APPLICATIONS
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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.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
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
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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.
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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
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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.
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OF EXEMPLARY EMBODIMENTS OF THE INVENTION
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.