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

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Title: 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. ...


Inventors: Dan M. Granoff, Peter Beernink, Jo Anne Welsch
USPTO Applicaton #: #20120107339 - Class: 4241901 (USPTO) - 05/03/12 - Class 424 
Drug, Bio-affecting And Body Treating Compositions > Antigen, Epitope, Or Other Immunospecific Immunoeffector (e.g., Immunospecific Vaccine, Immunospecific Stimulator Of Cell-mediated Immunity, Immunospecific Tolerogen, Immunospecific Immunosuppressor, Etc.) >Amino Acid Sequence Disclosed In Whole Or In Part; Or Conjugate, Complex, Or Fusion Protein Or Fusion Polypeptide Including The Same >Disclosed Amino Acid Sequence Derived From Bacterium (e.g., Mycoplasma, Anaplasma, Etc.)

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The Patent Description & Claims data below is from USPTO Patent Application 20120107339, Chimeric factor h binding proteins (fhbp) containing a heterologous b domain and methods of use.

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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.

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

This invention relates to vaccines for diseases caused by Neisseria meningitidis.

BACKGROUND

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.



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stats Patent Info
Application #
US 20120107339 A1
Publish Date
05/03/2012
Document #
File Date
07/24/2014
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