Modified Factor H Binding Proteins (FHBP) and methods of use thereof
阅读说明：本技术 改性的h因子结合蛋白（fhbp）及其使用方法 (Modified Factor H Binding Proteins (FHBP) and methods of use thereof ) 是由 P·T·伯尼克 D·M·格拉诺夫 R·帕乔菲特 于 2011-03-29 设计创作，主要内容包括：本申请涉及改性的H因子结合蛋白(FHBP)及其使用方法。本申请提供了H因子结合蛋白及其使用方法,所述蛋白能够引发针对至少一种脑膜炎奈瑟球菌菌株具有杀菌性的抗体。(The present application relates to modified Factor H Binding Proteins (FHBPs) and methods of use thereof. Factor H binding proteins and methods of use thereof are provided that are capable of eliciting antibodies that are bactericidal against at least one neisseria meningitidis strain.)
1. A non-naturally occurring factor H binding protein (fHbp) having lower affinity for human factor H (fH) than fHbp ID1, wherein said non-naturally occurring fHbp presents a conformational epitope to which an antibody binds, said antibody having bactericidal activity against at least one neisseria meningitidis strain.
2. The non-naturally occurring fHbp of claim 1, wherein said fHbp is derived from a variant 1fHbp, a variant 2fHbp, or a variant 3 fHbp.
3. The non-naturally occurring fHbp of claim 1, wherein said fHbp is a artificially prepared chimera, wherein said fHbp is derived from the amino acid sequence of chimera I, wherein said fHbp comprises an amino acid substitution at a position corresponding to amino acid residue 41.
4. An immunogenic composition comprising:
a) the fHbp of any one of claims 1-3; and
b) pharmaceutically acceptable adjuvants.
5. An immunogenic composition comprising:
a) an isolated factor H binding protein (fHbp) comprising at least 85% amino acid sequence identity to fHbp ID4, fHbp ID 14, fHbp ID15, wherein the fHbp has lower affinity for human factor H (fH) than fHbp ID 1; and
b) pharmaceutically acceptable adjuvants.
6. A nucleic acid encoding the fHbp of any one of claims 1-3.
7. A recombinant expression vector comprising the nucleic acid of claim 6.
8. A genetically modified host cell comprising the nucleic acid of claim 6 or the recombinant expression vector of claim 7.
9. A method of determining the likelihood that a factor H binding protein (fHbp) will elicit a bactericidal response in an individual against at least one neisseria meningitidis strain, the method comprising:
determining the ability of an antibody to inhibit binding of human factor H (fH) to fHbp, said antibody being present in serum obtained from an individual immunized with fHbp, wherein said antibody inhibits binding of fH to fHbp at a level at least about 25% greater than the level of inhibition of binding of fH to fHbp by a control antibody that inhibits binding of fH to fHbp but does not produce a bactericidal response, indicating that fHbp is likely to elicit a bactericidal response against at least one neisseria meningitidis strain.
10. A method of determining the likelihood that a non-naturally occurring factor H binding protein (fHbp) will elicit bactericidal antibodies in an individual against at least one neisseria meningitidis strain, the fHbp having lower affinity for human factor H (fH) than fHbp ID1, the method comprising:
determining the ability of antibodies elicited by a non-naturally occurring fHbp to inhibit binding of fH to fHbp in a non-human subject animal,
wherein the level of inhibition of binding of fH to fHbp by the antibodies elicited by the non-naturally occurring fHbp is at least about 25% greater than the level of inhibition of binding of fH to fHbp by the antibodies elicited by fHbp ID1 in the non-human subject animal, indicating that the non-naturally occurring fHbp is likely to elicit a bactericidal response against at least one neisseria meningitidis strain.
Summary of The Invention
Factor H binding proteins and methods of use thereof are provided that are capable of eliciting antibodies that are bactericidal against at least one strain of neisseria meningitidis.
The present application relates to the following.
1. A non-naturally occurring factor H binding protein (fHbp) having lower affinity for human factor H (fH) than fHbp ID1, wherein said non-naturally occurring fHbp presents a conformational epitope to which an antibody binds, said antibody having bactericidal activity against at least one neisseria meningitidis strain.
2. The non-naturally occurring fHbp of
3. The non-naturally occurring fHbp of
4. The non-naturally occurring fHbp of
5. The non-naturally occurring fHbp of
6. The non-naturally occurring fHbp of
7. The non-naturally occurring fHbp of
8. The non-naturally occurring fHbp of
9. The non-naturally occurring fHbp of
10. The non-naturally occurring fHbp of
11. The non-naturally occurring fHbp of
12. The non-naturally occurring fHbp of
13. The non-naturally occurring fHbp of
14. The non-naturally occurring fHbp of
15. The non-naturally occurring fHbp of
16. The non-naturally occurring fHbp of
17. The non-naturally occurring fHbp of
18. The non-naturally occurring fHbp of
19. The non-naturally occurring fHbp of item 17, wherein said fHbp comprises an amino acid substitution at a position corresponding to
20. The non-naturally occurring fHbp of
21. The non-naturally occurring fHbp of
22. The non-naturally occurring fHbp of item 17, wherein said fHbp comprises R130A as said amino acid substitution.
23. The non-naturally occurring fHbp of item 17, wherein said fHbp comprises H119A as said amino acid substitution.
24. The non-naturally occurring fHbp of item 17, wherein said fHbp comprises E218A or E239A as said amino acid substitution.
25. The non-naturally occurring fHbp of any one of claims 19-24, wherein said fHbp is derived from
26. The non-naturally occurring fHbp of item 17, wherein said fHbp comprises
27. The non-naturally occurring fHbp of item 17, wherein said fHbp comprises fHbp ID4, said fHbp ID4 containing R41S as said amino acid substitution.
28. The non-naturally occurring fHbp of item 17, wherein said fHbp comprises fHbp ID9, said fHbp ID9 containing R41S as said amino acid substitution.
29. The non-naturally occurring fHbp of item 17, wherein said fHbp comprises fHbp ID74, said fHbp ID74 containing R41S as said amino acid substitution.
30. The non-naturally occurring fHbp of item 17, wherein said fHbp is fHbp ID15, said fHbp ID15 containing S41P as said amino acid substitution.
31. The non-naturally occurring fHbp of item 17, wherein said fHbp comprises R80A as said amino acid substitution.
32. The non-naturally occurring fHbp of item 17, wherein said fHbp comprises D211A as said amino acid substitution.
33. The non-naturally occurring fHbp of item 17, wherein said fHbp comprises E218A as said amino acid substitution.
34. The non-naturally occurring fHbp of item 17, wherein said fHbp comprises E248A as said amino acid substitution.
35. The non-naturally occurring fHbp of item 17, wherein said fHbp comprises G236I as said amino acid substitution.
36. The non-naturally occurring fHbp of item 17, wherein said fHbp comprises an amino acid substitution at one or both of positions 220 and 222.
37. The non-naturally occurring fHbp of item 17, wherein said fHbp comprises T220A and H222A as said amino acid substitutions.
38. The non-naturally occurring fHbp of any one of claims 31-37, wherein said fHbp is derived from
39. The non-naturally occurring fHbp of item 17, wherein said fHbp comprises an amino acid substitution at one or more of
40. The non-naturally occurring fHbp of item 17, wherein said fHbp comprises R41S and K113A as said amino acid substitutions.
41. The non-naturally occurring fHbp of item 17, wherein said fHbp comprises R41S and K119A as said amino acid substitutions.
42. The non-naturally occurring fHbp of
43. The non-naturally occurring fHbp of
44. The non-naturally occurring fHbp of any one of claims 39-43, wherein said fHbp is derived from
45. The non-naturally occurring fHbp of item 17, wherein said fHbp is derived from the amino acid sequence of chimera I and comprises R41S as the amino acid substitution.
46. The non-naturally occurring fHbp of item 17, wherein said fHbp is fHbp ID28, said fHbp ID28 comprising E218A and/or K199A as said amino acid substitution.
An immunogenic composition comprising:
a) the fHbp of any one of claims 1-46; and
b) pharmaceutically acceptable adjuvants.
48. An immunogenic composition comprising:
a) an isolated factor H binding protein (fHbp) comprising at least 85% amino acid sequence identity to fHbp ID4,
b) pharmaceutically acceptable adjuvants.
49. The immunogenic composition of
50. The immunogenic composition of
51. The immunogenic composition of
52. A method of eliciting an antibody response in a mammal, the method comprising administering to a mammal a fHbp according to any of items 1-46, or an immunogenic composition according to
53. The method of item 52, wherein the mammal is a human.
54. The method of item 52, wherein the administering is for the production of bactericidal antibodies against Neisseria meningitidis.
55. A nucleic acid encoding a fHbp according to any one of items 1-46.
56. A recombinant expression vector comprising the nucleic acid of
57. A genetically modified host cell comprising the nucleic acid of
58. An immunogenic composition comprising:
a) a vesicle from a genetically modified neisseria host cell, the cell being genetically modified with a nucleic acid encoding the fHbp of any one of items 1-46, such that the genetically modified host cell produces the encoded non-naturally occurring fHbp, wherein the vesicle comprises the encoded non-naturally occurring fHbp; and
b) pharmaceutically acceptable adjuvants.
59. The immunogenic composition of
60. The immunogenic composition of
61. The immunogenic composition of
62. The immunogenic composition of
63. A method of eliciting an antibody response in a mammal, the method comprising administering to a mammal the immunogenic composition of
64. A method of determining the likelihood that a factor H binding protein (fHbp) will elicit a bactericidal response in an individual against at least one neisseria meningitidis strain, the method comprising:
determining the ability of an antibody to inhibit binding of human factor H (fH) to fHbp, said antibody being present in serum obtained from an individual immunized with fHbp, wherein said antibody inhibits binding of fH to fHbp at a level at least about 25% greater than the level of inhibition of binding of fH to fHbp by a control antibody that inhibits binding of fH to fHbp but does not produce a bactericidal response, indicating that fHbp is likely to elicit a bactericidal response against at least one neisseria meningitidis strain.
65. The method of item 64, wherein the fHbp is a non-naturally occurring fHbp having a lower affinity for fH than
66. A method of determining the likelihood that a non-naturally occurring factor H binding protein (fHbp) will elicit bactericidal antibodies in an individual against at least one neisseria meningitidis strain, the fHbp having a lower affinity for human factor H (fH) than fHbp ID1, the method comprising:
determining the ability of antibodies elicited by a non-naturally occurring fHbp to inhibit binding of fH to fHbp in a non-human subject animal,
wherein the level of inhibition of binding of fH to fHbp by the antibodies elicited by the non-naturally occurring fHbp is at least about 25% greater than the level of inhibition of binding of fH to fHbp by the antibodies elicited by fHbp ID1 in the non-human subject animal, indicating that the non-naturally occurring fHbp is likely to elicit a bactericidal response against at least one neisseria meningitidis strain.
Brief Description of Drawings
FIG. 1: panel a, standard curve of human fH concentration measured by ELISA with meningococcal fHbp as antigen in each well. See example 1 for details. Panel B, concentration of human fH in serum of human fH transgenic (Tg) mice, including human fH-Tg negative littermates or known wild-type BALB/c mice, and human fH concentration in human serum. See example 1.
FIG. 2: serum IgG antibody responses of human fH transgenic (fH Tg) BALB/C mice and wild-type (WT) BALB/C mice (panels a and B), and serum bactericidal titers against group C4243 strains (panel C) following immunization with group C meningococcal conjugate control vaccine. The conjugate vaccine did not bind human fH. See example 1 for details. Small graph D: human fH binds to the wild-type fHbp vaccine but not to the control MenC-CRM conjugate vaccine or certain mutant fHbp vaccines, as shown in the corresponding figures in table 5 of the examples section. Mouse (or rabbit, or rat, etc.) fH does not bind to wild-type fHbp.
FIG. 3: the concentration of human fH in serum of fH transgenic mice was correlated with serum bactericidal antibody response following vaccination with either wild-type fHbp that binds human fH (panel a) or R41S mutant that does not bind human fH (panel B). Panel C is the GMT ratio (correlation of mutant/wild type vaccine to human fH serum concentration in immunized fH transgenic mice) estimated using a general linear regression model. See example 4 for details.
FIG. 4: binding of human fH, anti-fHbp mAb, JAR4, and JAR5 to wild-type and mutant fHbp (mutant of fHbp id1, containing Glu replaced by Ala) was determined using enzyme-linked immunosorbent assay (ELISA).
FIG. 5: SDS-PAGE size and purity analysis of WT fHbp ID1 and ID1 double mutant E218A/E239A. Molecular weights in kDa are shown on the left.
FIG. 6: soluble fHbp inhibition of anti-fHbp MAb binding to immobilized wild-type fHbp was detected by ELISA.
FIGS. 7A-7D: figures 7A and 7B show differential scanning calorimetry results for the wild-type fHbp ID1 and E218A/E239A double mutant protein (figure 7A) and the wild-type fHbp ID1 and R41S mutant protein (figure 7B). FIG. 7C shows anti-fHbp IgG antibody titer of mice determined by ELISAAnd (c) immunizing the mouse with the wild-type fHbp ID1 or E218A/E239A double mutant protein. IgG anti-fHbp antibody responses in mice immunized with WT or mutant fHbp. In
FIG. 8: panel a shows binding of fH to the native fHbp variant. Wells of the elisa plate were coated with recombinant fHbp representing variant fHbp ID1, 14, or 15. Binding of human fH was detected as described in the examples section. Panels B and C show binding of variants fHbp ID1, 14, and 15 to Mab JAR4 and JAR5, respectively.
FIGS. 9A-9B: fig. 9A, structure of fHbp complexed with human fH fragment. In the cartoon representation, fHbp is indicated in black at the bottom and fH in grey at the top. The structural model of fHbp binding to fH fragments is based on published atomic coordinates (Schneideret al. ((2009) Nature 458: 890-3)). The black bands represent the N-and C-terminal domains of the fHbp molecules, respectively. The grey bands represent the sixth and seventh short homologous repeat domains of human fH previously published that mediate the interaction of human fH with fHbp (Schneideret al ((2009) Nature 458: 890-3). the partial enlargement on the left is the arginine residue at
FIG. 10: binding of fH (panel a) or anti-fHbp Mab JAR4 (panel B) or JAR5 (panel C) to R41S and R41A mutants of fHbp ID1 detected by ELISA. The binding detection of human fH (panel a) and anti-fHbp MAb (panels B and C) is described in example 2.
FIG. 11: binding of human factor H (left column) or anti-fHbp Mab JAR4 (right column) to different fhbps in
FIG. 12: binding of human fH and anti-fHbp MAb controls to the corresponding R41S mutant of fHbp and fHbp in
FIG. 13: effect of anti-fHbp antibodies elicited in serum of human fH transgenic mice on binding of fH to fHbp. Binding of fH to fHbp was detected by ELISA using 1:100 dilutions of pre-immune (panel a, pre-immune) and post-immune (panel B, post-immune) sera from each transgenic mouse immunized with the wild-type fHbp ID1 or R41S mutant ID 1fHbp vaccine. For the aluminum control group, open squares represent data from pooled sera from transgenic mice containing human fH, and closed triangles represent data from sera from wild-type mice that do not contain human fH. OD values represent the amount of bound human fH detected using sheep anti-human fH and donkey anti-sheep IgG coupled to alkaline phosphatase. Panel C, anti-fHbp IgG titers in serum after immunization indicate that similar antibody responses were generated against both vaccines. Panel D, inhibition of binding of human fH to fHbp in the presence of added human fH. Panel E, percent inhibition of fH binding as a function of SBA titer in human fH transgenic mice immunized with fHbp vaccines.
FIG. 14: panels a and B show binding of the K241E mutant of fHbp ID1 to fH and MAb JAR5, respectively. Panels C and D show binding of E241K mutant of fHbp ID15 to fH and MAb JAR5, respectively. Binding of fH or anti-fHbp MAb to fHbp was detected as described in example 2.
FIG. 15: binding of fH or anti-fHbp MAb to single mutants of H119A and R130A of fHbp ID1 was detected by ELISA. Binding of human fH (panel a) to anti-fHbp MAb JAR5 (panel B) or JAR4 (panel C) was tested as described in example 2.
FIG. 16 schematic representation of the six most common modular groups of fHbp, designated I through VI, the variable fragments were derived from one of two genetic lineages, designated α (in grey) or β (in white), according to the nomenclature adopted by pubmlst. org/neisseria/fHbp/website, the α and β lineages also can be designated
FIG. 17: binding of fH to recombinant fHbp mutant S41P (mutant of fHbp ID 15). Panel a shows binding of fH to S41P mutant of
FIG. 18: binding of human fH to the R41S mutant of fHbp chimera I (Beernink et al (2008) Infec. Immun.76:2568-2575) (panel A) and to the corresponding JAR5 (panel B).
FIG. 19, Panel A, alignment of natural variants with the fHbp sequence of an artificial chimera (chimera I; Beernik et al (2008) Infec. Immun.76: 2568-2575.) fHbp ID1 belongs to modular group I (all five variable fragments, A-E, all derived from the α lineage) according to the definition of Beernik and Granoff (2009) Microbiology155: 2873-83. fHbp ID28 belongs to modular group II (all five fragments are derived from the β lineage). fHbp ID15 is a β -type A fragment (V) of a natural chimera (belongs to modular group IV, having one β A fragment and α B, C, D, and E fragment) fHbp ID28A(ii) a Residues 8-73) and the corresponding A fragment of fHbp ID15 (V)A) In contrast, the latter also has β -type A fragment (V)Aβ). rectangle indicates the residues that are changed in the E218A/E239A double mutation fHbp.Chart B, alignment of fHbp ID1 and the A fragment of fHbp ID77 (
FIG. 20: binding of fH or anti-fHbp MAb to the K113A, K119A, and D121A single mutants of fHbp ID77 was tested by ELISA. Binding of human fH (panel a) and anti-fHbp MAb JAR31 (panel B) to fHbp ID77 mutants was tested as described in example 2.
FIG. 21: binding of fH or anti-fHbp MAb to the double mutants of R41S/K113A, R41S/K119A, and 41S/D121A of fHbp ID77 was tested by ELISA. Binding of human fH (panel a) and anti-fHbp MAb JAR31 (panel B) to fHbp ID77 mutants was tested as described in example 2.
FIG. 22: binding of fH or anti-fHbp MAb to the K113A/D121A double mutant and R41S/K113A/D121A triple mutant of fHbp ID77 was tested by ELISA. Binding of human fH (panel a), anti-fHbp MAb JAR4 (panel B) and anti-fHbp MAb JAR31 (panel C) to fHbp ID77 mutants was tested as described in example 2.
FIG. 23: binding of fH to fHbp ID22 mutant was detected by ELISA. Binding of human fH to D221A, R80A, or wild-type fHbp was detected as described in example 2 (panel a), binding of human fH to E218A, E248A, or wild-type fHbp (panel B), and binding of human fH to R41S, Q38A, Q126A, or wild-type fHbp (panel C).
FIG. 24: binding of anti-fHbp MAb JAR31 to fHbp ID22 mutant was detected by ELISA. Binding of the R80A and D211A mutants to JAR31 was tested as described in example 2 (panel a) and binding of the E218A and E248A mutants to JAR31 (panel B).
FIG. 25: binding of anti-fHbp Mab JAR4 to fHbp ID22 mutant was detected by ELISA. Binding of the R80A and D211A mutants to JAR4 was tested as described in example 2 (panel a) and binding of the E218A and E248A mutants to JAR4 (panel B).
FIG. 26: binding of anti-fHbp MAb JAR35 to fHbp ID22 mutant was detected by ELISA. Binding of the R80A and D211A mutants to JAR35 was tested as described in example 2 (panel a), and binding of the E218A and E248A body mutations to JAR35 (panel B).
FIG. 27 is a schematic view showing: the binding of anti-fHbp MAb to the T220A/H22A double mutant of fHbp ID22, or the G236I mutant was tested by ELISA. Binding of human fH (panel a), anti-fHbp MAb JAR31 (panel B), JAR35 (panel C), or JAR4 (panel D) to fHbp ID22 mutants was tested as described in example 2.
FIG. 28: binding of fH or anti-fHbp MAb to R41S, Q38A, and a235G mutants of fHbp ID22 was tested by ELISA. Binding of human fH (panel a), anti-fHbp MAb JAR31 (panel B) or JAR35 (panel C) to fHbp ID22 mutants was tested as described in example 2.
FIG. 29: binding of fH or anti-fHbp MAb to Q126A, D201A, and E202A mutants of fHbp ID22 was tested by ELISA. Binding of human fH (panel a), anti-fHbp MAb JAR35 (panel B) and fHbp ID22 mutants was tested as described in example 2.
FIG. 30: binding to fHbp ID28 (variant group 3) mutant. Panels a and C, binding of fH to K199A, E217A, and E218A mutants was detected using ELISA. Panels B and D show the binding of anti-fHbp MAb JAR31 (panel B), and anti-fHbp MAb JAR33 (panel D, only fHbp wild-type ID28 WT and E218A mutants) to fHbp ID28 mutants.
FIG. 31 depicts serum bactericidal titers of wild-type BLAB/c mice immunized with the indicated mutants of the fHbp ID1 vaccine and measured with titers against serogroup B strain H44/76(fHbp ID 1).
Figure 32 depicts serum bactericidal titers of mice immunized with the indicated mutant of fHbp ID22 and tested with
FIG. 33 depicts bactericidal titers in mice immunized with the R41S/K113A/D121A triple mutant of fHbp ID77 and tested for titer against serogroup W-135
FIG. 34: alignment of the amino acid sequences of fHbp ID 1(SEQ ID NO:1), fHbp ID22 (SEQ ID NO:2), fHbp ID77 (SEQ ID NO:4), fHbp ID28 (SEQ ID NO:3), and the ID1/ID77 chimera (SEQ ID NO: 8). ID28 served as a control sequence for
FIG. 35: alignment of the amino acid sequences of fHbp ID 1(SEQ ID NO:1), fHbp ID22 (SEQ ID NO:2), fHbp ID77 (SEQ ID NO:4), fHbp ID28 (SEQ ID NO:3), and the ID1/ID77 chimera (SEQ ID NO: 8). Residues marked in grey represent mutated residues, which are summarized in table 7.
Figure 36 depicts a model of fHbp in complexes formed with fH fragments. The positions of amino acid residues known to affect the epitopes of
FIGS. 37A-37D depict the binding of human IgGa mouse chimeric fHbp-specific mAb to fHbp detected by ELISA (FIG. 37A), plasmon resonance (FIG. 37B), flow cytometry against live bacteria (FIG. 37C, mAb only; and FIG. 37D, mAb in the presence of 20% IgG depleted human serum).
FIGS. 38A-38B depict the C1 q-dependent C4B deposition of human IgG1 mouse chimeric anti-fHbp mAbs JAR3, JAR5 and mAb502, activated complement on enveloped, serogroup B strain H44/76 bacteria. FIG. 38A, human serum with C1q removed as a source of complement; FIG. 38B, purified C1q protein was added to C1 q-depleted serum prior to reaction. Figure 38C depicts the bactericidal activity of each mAb mediated by human complement, tested against serogroup B strain H44/76.
FIGS. 39A-39C depict the attachment of fHbp to wells of a microplate and the inhibition of fH binding by anti-fHbp mAb by ELISA (FIG. 39A), and by flow cytometry using live bacteria of serogroup B strain H44/76 (FIGS. 39B and 39C).
Figures 40A-40C depict binding of fH to serogroup B H44/76 mutants that genetically inactivate fHbp expression, or both fHbp and NspA. FIG. 40A, binding of control anti-PorA mAb; FIGS. 40B and 40C, binding of fH in human serum from which IgG has been removed.
FIGS. 41A-41E depict the bactericidal activity of human IgG mouse chimeric anti-fHbp mAb, detected against NspA genetically inactivated serogroup B H44/76 mutant. Fig. 41A, 41B, and 41C: anti-fhbp mabs JAR3, JAR5 and mAb502, respectively; fig. 41D and 41E: control anti-PorA and anti-capsular mAb, respectively.
Fig. 42 depicts bactericidal activity of anti-NspA antibodies against capsular group a strain (
Figure 43, panels a-C depict serum anti-fHbp antibody responses of wild-type mice immunized with recombinant fHbp vaccines or natural outer membrane vesicle vaccines from fHbp over-expressed or fHbp knock-out serogroup B strain H44/76 mutants. The anti-fHbp antibody response to immunization was measured by ELISA (panel a), or the ability of serum anti-fHbp antibodies to inhibit fH binding to fHbp (panels B and C, also measured by ELISA). Mice were immunized with either recombinant fHbp ID1 vaccine (closed triangles), or NOMV vaccine from either fHbp ID1 over-expressed (open circles) or fHbp knock-out (closed circles) serogroup B strain H44/76 mutants.
FIG. 44 shows the amino acid sequence of a Neisseria surface protein A (NspA) polypeptide (SEQ ID NO: 15).
FIG. 45: amino acid sequence of various naturally occurring factor H binding proteins (fHbp): fHbp ID1, fHbp ID15, fHbp ID22, fHbp ID28, fHbp ID77, and chimera I (Beernink et al (2008) Infec. Immun.76: 2568-. FHBP ID sequence shown has no leader sequence. In the sequence of chimera I, the lower case letters correspond to the amino acid sequence derived from fHbp ID1, and the upper case letters correspond to the amino acids derived from
Before the present invention and certain exemplary embodiments thereof 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.
When referring to a range of values, it is understood that the invention includes every 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. The invention also includes the upper and lower limits of these smaller ranges, which may independently be included in the smaller ranges, except where expressly excluded in the stated ranges. Where the stated range includes one or both of the limits, the range excluding those included limits is also included in the invention.
For clarity, certain features of the invention are described in the context of separate embodiments, and it is to be understood that these features may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of embodiments involving amino acid modifications, including amino acid substitutions, as compared to a reference amino acid sequence are specifically included and disclosed herein as if each combination were independently and explicitly disclosed, so long as such combinations comprise a polypeptide having the desired characteristics, e.g., a non-naturally occurring fHbp polypeptide having a lower affinity for human fH than
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 described below. 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 in this application and 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, reference to "the protein" includes reference to one or more proteins, and the like.
The publications discussed in this application are provided solely for their disclosure prior to the filing date of the present application. Nothing in this application should be construed as an admission that the 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
Factor H binding proteins and methods of use thereof are provided that are capable of eliciting antibodies that are bactericidal against at least one neisseria meningitidis strain.
Definition of
"factor H binding proteins" (fHbp), also referred to in the literature as GNA1870, ORF2086, LP2086 (lipoprotein 2086), and "741", refer to a class of neisseria meningitidis polypeptides. It is found in nature to be a lipoprotein on the surface of bacteria. Based on the variability of amino acid sequence and immunological cross-reactivity (Masignani et al (2003) J Exp Med197:789-99), N.meningitidis strains have been subdivided into three groups of fHbp variants (referred to as variant 1(v.1), variant 2(v.2) and variant (v.3) in some reports (Masignani et al (2003) J Exp Med197:789-99) and as families A and B in other reports (see, e.g., Fletcher et al (2004) Infect Immun72: 2088-2100)). Each unique fHbp found in neisseria meningitidis was assigned a fHbp peptide ID according to neisseria org or pubmlst org/neisseria/fHbp/website. Because the length of the variant 2(v.2) fHbp proteins (from strain 8047, fHbp ID 77) and the variant 3(v.3) fHbp (from strain M1239, fHbp ID 28) differ from the length of MC58(fHbp ID 1) by-1 and +7 amino acid residues, respectively, the numbering used to refer to v.2 and v.3fHbp protein residues differs from the numbering based on the actual amino acid sequence of these proteins. Thus, for example, the leucine residue (L) at position 166 of the v.2 or v.3fhbp sequence corresponds to residues 165 and 173 of the v.2 and v.3 proteins, respectively.
Human factor H ("human fH"), as used herein, refers to a protein comprising the amino acid sequence shown in FIG. 9B (SEQ ID NO: 9), and naturally occurring human allelic variants thereof.
For example, where "heterologous" is used in the context of a chimeric polypeptide, the chimeric polypeptide comprises operably linked amino acid sequences from different phylogenetic groups of different polypeptides (e.g., a first component derived from an α progenitor amino acid sequence and a second component derived from an β progenitor amino acid sequence). the chimeric polypeptide contains two or more defined fragments, each fragment being from a different ancestor, respectively, and can be a naturally occurring or artificially prepared (non-naturally occurring) polypeptide.
A "heterologous" or "chimeric" polypeptide can also comprise two or more different components, each component being from a different fHbp (e.g.,
"heterologous" in the context of a polynucleotide encoding any chimeric polypeptide as described above includes operably linked nucleic acid sequences from different genes (e.g., a first component derived from a nucleic acid encoding a fHbp v.1 polypeptide and a second component derived from a nucleic acid encoding a fHbp v.2 polypeptide) or nucleic acid sequences from different progenitor amino acid sequences (α or β).
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 genetically different from the coding sequence (e.g., the host cell may be genetically different from the promoter, coding sequence, or both, in order to provide for expression in the host cell of interest). For example, when the T7 promoter is operably linked to a polynucleotide encoding a fHbp polypeptide or domain thereof, the T7 promoter is said to be a heterologous nucleic acid.
"heterologous" in the context of a recombinant cell refers to the presence in the host cell of a nucleic acid (or gene product, such as a polypeptide) that is not genetically derived from it. For example, the amino acid or nucleic acid sequence of one neisserial strain is heterologous to the host of another neisserial strain.
"derived from" in the context of an amino acid sequence or polynucleotide sequence (e.g., "derived from" the amino acid sequence of fHbp ID 1) is used to indicate that the sequence of the polypeptide or nucleic acid is based on the sequence of a control polypeptide or nucleic acid (e.g., a naturally occurring fHbp protein or encoding nucleic acid) and is not intended to limit the source or method of making the protein or nucleic acid. Non-limiting examples of control polypeptides and control polynucleotides from which an amino acid sequence or polynucleotide sequence may be "derived" include naturally occurring fHbp, fHbp ID1, and non-naturally occurring fHbp. "derived from" in the context of a bacterial strain is used to indicate a strain obtained by in vivo or in vitro subculture of a parent strain, and/or a recombinant cell obtained by modifying a parent strain.
"conservative amino acid substitution" refers to the substitution of one amino acid residue in the side chain of an amino acid with another amino acid having similar chemical and physical properties (e.g., charge, size, hydrophobicity/hydrophilicity). "conservative substitutions" are intended to include substitutions within the following group of amino acid residues: gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr; lys, arg; and phe, tyr. Such substitutions are directed by aligning the amino acid sequences of the polypeptides in which the epitope of interest is present.
The term "protective immunity" refers to the administration of a vaccine or immunization regimen to a mammal to induce an immune response that prevents, delays the development of, or reduces the severity of, or reduces or altogether eliminates the symptoms of a disease caused by neisseria meningitidis. Protective immunity may be accompanied by the production of bactericidal antibodies. It should be noted that it is well recognized in the art that the production of bactericidal antibodies against neisseria meningitidis predicts that the vaccine has a protective effect in humans (Goldschneider et al (1969) j.exp.med.129: 1307; Borrow et al (2001) infectimmun.69: 1568).
The phrase "a disease caused by a neisseria meningitidis strain" includes any clinical symptom or combination of clinical symptoms exhibited by infection of neisseria meningitidis in humans. These symptoms include, but are not limited to: neisseria meningitidis pathogenic strains colonize the upper respiratory tract (e.g., nasopharynx and tonsillar mucosa), bacteria enter mucosal and submucosal vascular beds, sepsis, septic shock, inflammation, hemorrhagic skin damage, activation of fibrinolysis and blood clotting, organ dysfunction, such as kidney, lung and heart failure, adrenal bleeding and muscle infarction, capillary leakage, edema, peripheral limb ischemia, respiratory distress syndrome, pericarditis and meningitis.
In the context of antigens (e.g., polypeptide antigens), the phrase "specifically binds to an antibody" or "specifically immunoreactive" refers to a binding reaction based on and/or tested for the presence of an antigen in a sample that may contain a heterogeneous population of other molecules. Thus, under the specified conditions, the one or more specific antibodies are capable of binding to one or more specific antigens in the sample, but not to a substantial extent to other molecules present in the sample. An epitope of an antigen (e.g., an epitope of a polypeptide) "specifically binds" or "specifically immunoreacts" with an antibody refers to the binding reaction based on and/or testing for the presence of the epitope in the antigen (e.g., polypeptide) in a heterogeneous population that may contain other epitopes, and in a heterogeneous population that may contain other antigens. Thus, under the conditions specified, the specific antibody or antibodies are capable of binding to a particular epitope in the antigen and not to a significant extent to other epitopes present in the antigen and/or sample.
The phrase "in an amount sufficient to elicit an immune response" means that there is a detectable difference between the indicators of the immune response detected before and after administration of a particular preparation of antigen. Indicators of immune response include, but are not limited to, antibody titer or specificity as detected by: enzyme linked immunosorbent assay (ELISA), bactericidal assay, flow cytometry, immunoprecipitation, Ouchterlony immunodiffusion; binding detection assays such as spots, Western blots, or antigen arrays; cytotoxicity tests, etc.
A "surface antigen" is an antigen that is present in the surface structure (e.g., outer membrane, capsule, pilus, etc.) of Neisseria meningitidis.
By "isolated" is meant that the environment of the compound of interest is different from its naturally occurring environment. "isolated" is intended to include compounds in a sample that are substantially enriched in a compound of interest, and/or in which the compound of interest is partially or substantially purified.
"enrichment" refers to subjecting a sample (e.g., by an experimenter or clinician) to a non-natural manipulation that results in a concentration of a compound of interest that is greater (e.g., at least 3-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 a starting sample, e.g., a biological sample (e.g., a sample in which the compound naturally occurs or is present after administration) or a sample in which the compound is prepared (e.g., as in bacterial polypeptides, antibodies, chimeric polypeptides, etc.).
A target gene "knockout" refers to a change in gene sequence that results in a decrease in the function of the target gene, e.g., results in undetectable or insignificant expression of the target gene, and/or no or significant function of the gene product. For example, after "knocking out" a gene involved in LPS synthesis, gene function is greatly reduced, resulting in undetectable, or only insignificant levels of gene expression, and/or significantly reduced or undetectable biological activity (e.g., enzymatic activity) of the gene product compared to prior to modification. "knockdown" includes conditional knockdown, for example, where alteration of a target gene can occur when exposed to a predetermined condition (e.g., temperature, osmotic pressure), and when exposed to a substance that promotes alteration of the target gene, or the like. A target gene "knockin" refers to a genetic alteration in a gene that results in an increased function of the target gene.
FHBP polypeptides with altered FH binding properties
Before describing more fHbp that the present invention is intended to disclose, it is helpful to describe some of the naturally occurring fHbp. Each unique and naturally occurring fHbp found in neisseria meningitidis was assigned a fHbp peptide ID as shown by the websites of neisseria org and pubmlst org/neisseria/fHbp. This fHbp naming convention will be employed in the present invention.
For convenience and clarity, the native amino acid sequence of fHbp ID1 (v.1fhbp of neisseria meningitidis strain MC 58) was selected as a control sequence for all naturally occurring and non-naturally occurring fHbp amino acid sequences in this application, including fHbp chimeras and/or variants described herein. The amino acid sequence of fHbp ID1 is shown in figure 45 and below:
fHbp ID1
CSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKRQFRIGDIAGEHTSFDKLPEGGRATYRGTAFGSDDAGGKLTYTIDFAAKQGNGKIEHLKSPELNVDLAAADIKPDGKRHAVISGSVLYNQAEKGSYSLGIFGGKAQEVAGSAEVKTVNGIRHIGLAAKQ(SEQ ID NO:1)。
the position numbers used in the present application when referring to the position of the amino acid residue in fHbp correspond to the amino acid residue numbers of
The present application provides fHbp, compositions comprising fHbp, and methods of using the fHbp and compositions. The fHbp has a lower affinity for human fH than a corresponding control fHbp (e.g., a naturally occurring fHbp; or other control fHbp). Because of the high affinity of fHbp in its complex with fH, binding of fH masks one or more epitopes on fHbp from recognition by the host immune system. Thus, fHbp complexed and/or bound to fH may not be as effective an immunogen as uncomplexed fHbp. Conversely, fHbp having a relatively low affinity for fH when administered as an immunogen (e.g., in a vaccine composition) is capable of presenting certain epitopes to the immune system of the immunized host that are not presented by fHbp having a high affinity for fH, as binding to fH may mask such epitopes. The fHbp has a low affinity for human fH and is useful for eliciting bactericidal antibodies against neisseria meningitidis and/or for providing protective immunity against neisseria meningitidis. The fHbp is a non-naturally occurring fHbp. Non-naturally occurring fHbp, which cannot be found in nature, is prepared manually, and/or is intentionally modified manually. The non-naturally occurring fHbp can be prepared by chemical synthesis or recombinant methods.
As used herein, "low affinity," "lower affinity," or "fH low binding protein" means that fHbp binds human fH with as low an affinity as fHbp ID1 or less than
The low affinity fHbp binds human fH with no more than about 100%, no more than about 95%, no more than about 90%, no more than about 85%, no more than about 80%, no more than about 75%, no more than about 70%, no more than about 65%, no more than about 60%, no more than about 50%, no more than about 45%, or with less than the affinity of the high affinity fHbp (e.g., fHbp ID 1) for human fH. For example, the subject fHbp has less affinity for human fH than about 50% of
In certain embodiments, the subject non-naturally occurring fHbp binds human fH with a binding affinity that is 85% or less of the binding affinity of wild-type fHbp to human fH. For example, in certain embodiments, the binding affinity of a subject non-naturally occurring fHbp to human fH is about 85% to about 75%, about 75% to about 65%, about 65% to about 55%, about 55% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1% of the binding affinity of wild-type fHbp to human fH. For example, in certain embodiments, the binding affinity of a subject's non-naturally occurring fHbp to human fH is about 85% to about 75%, about 75% to about 65%, about 65% to about 55%, about 55% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1% of the binding affinity of fHbp ID1 to human fH.
For example, in certain embodiments, a mutant of subject fHbp ID1 (e.g., R41S, R41A, R130A, H119A, E218A, or E239A mutant of fHbp ID 1) binds human fH with a binding affinity that is about 85% to about 75%, about 75% to about 65%, about 65% to about 55%, about 55% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1% of the binding affinity of fHbp ID1 to human fH.
For another example, in certain embodiments, the subject fHbp ID4, ID9, or mutant of ID74 (e.g., fHbp ID4, ID9, or R41S mutant of ID 74) has a binding affinity to human fH of from about 85% to about 75%, from about 75% to about 65%, from about 65% to about 55%, from about 55% to about 45%, from about 45% to about 35%, from about 35% to about 25%, from about 25% to about 15%, from about 15% to about 10%, from about 10% to about 5%, from about 5% to about 2%, from about 2% to about 1%, or from about 1% to about 0.1%, or less than 0.1% of the binding affinity of fHbp ID4, ID9, or ID74 to human fH, or the binding affinity of fHbp ID1 to human fH.
For another example, in certain embodiments, the binding affinity of a mutant of subject fHbp ID22 (e.g., R80A, D211A, E218A, E248A, G236I, or T220A/H222A mutant of fHbp ID 22) to human fH is about 85% to about 75%, about 75% to about 65%, about 65% to about 55%, about 55% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1% of the binding affinity of fHbp ID22 to human fH, or of fHbp ID1 to human fH.
For another example, in certain embodiments, a mutant of subject fHbp ID77 (e.g., R41S/K113A, R41S/K119A, R41S/D121A, or R41S/K113A/D121A mutant of fHbp ID 77) binds human fH with a binding affinity of fHbp ID77 to human fH, or with a binding affinity of fHbp ID1 to human fH of about 85% to about 75%, about 75% to about 65%, about 65% to about 55%, about 55% to about 45%, about 45% to about 35%, about 35% to about 25%, about 25% to about 15%, about 15% to about 10%, about 10% to about 5%, about 5% to about 2%, about 2% to about 1%, or about 1% to about 0.1%, or less than 0.1%.
For another example, in certain embodiments, the binding affinity of a mutant of subject fHbp ID28 (e.g., E218A mutant of
The term dissociation constant (K) can be usedD) Binding affinity is described. Dissociation constant (K) of low affinity fHbp from human fHD(ii) a M) is at least greater than about 80%, at least greater than about 100%, at least greater than about 120%, at least greater than about 140%, at least greater than about 160%, at least greater than about 200%, or greater than the K of high affinity fHbp (e.g., fHbp ID 1) and human fHD. K of low affinity fHbpDK described as fHbp ID1DAbout 2X (2 times), about 3X, about 5X ofAbout 10X, about 15X, about 20X, up to about 50X or more.
As used herein, "having a lower affinity for human fH than the corresponding fHbp" is used to describe fhbps that bind with a lower affinity than the corresponding control fHbp.
In many cases, the corresponding fHbp for comparing subject fHbp binding affinities ("control f' Hbp") is
The amino acid sequences of some examples of naturally occurring fHbp and artificially made chimeras are shown below and in figure 45.
CSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPSGKAEYHGKAFSSDDPNGRLHYSIDFTKKQGYGRIEHLKTPEQNVELASAELKADEKSHAVILGDTRYGGEEKGTYHLALFGDRAQEIAGSATVKIREKVHEIGIAGKQ(SEQ ID NO:2)
CSSGGGGSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDSIPQNGTLTLSAQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQNHSAVVALQIEKINNPDKTDSLINQRSFLVSGLGGEHTAFNQLPGGKAEYHGKAFSSDDPNGRLHYSIDFTKKQGYGRIEHLKTLEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ(SEQ ID NO:3)
CSSGGGGVAADIGARLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ(SEQ ID NO:4)
CSSGGGGSGGGGVAADIGAGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAERTFKAGDKDNSLNTGKLKNDKISRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQTEQVQDSEHSGKMVAKRQFRIGDIVGEHTSFGKLPKDVMATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAAADIKPDEKHHAVISGSVLYNQAEKGSYSLGIFGGQAQEVAGSAEVETANGIRHIGLAAKQ(SEQ ID NO:5)
FHbp ID 6
CSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVNGQLITLESGEFQVYKQSHSALTALQTEQVQDSEHSRKMVAKRQFRIGDIAGEHTSFDKLPKGDSATYRGTAFGSDDAGGKLTYTIDFAAKQGYGKIEHLKSPELNVDLAAAYIKPDEKHHAVISGSVLYNQDEKGSYSLGIFGGQAQEVAGSAEVKTANGIRHIGLAAKQ(SEQ ID NO:6)
CSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQTEQEQDPEHSGKMVAKRRFKIGDIAGEHTSFDKLPKDVMATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVELATAYIKPDEKHHAVISGSVLYNQDEKGSYSLGIFGGQAQEVAGSAEVETANGIHHIGLAAKQ(SEQ ID NO:7)
Chimera I
The corresponding fHbp may be naturally occurring and/or non-naturally occurring (e.g. artificially prepared chimeric) fHbp from which the subject fHbp is derived, naturally occurring chimeric fHbp having variable fragments derived from different progenitors (α or β) as a result of which the molecular architecture is shown to be modular, fHbp variants may be further classified in modular groups according to different combinations of five variable fragments, each variant derived from one of two genetic lineages named α -or β -type (Pajon R et al (2010) Vaccine28: 2122-9; berning k, Granoff DM (2009) Microbiology155: 2873-83). the six modular groups named I to VI may encompass > 95% (Pajon R et al (2010) Vaccine28:2122-9) of all known fHbp variants, the framework of the naturally occurring reference hbp group 16.
The corresponding fHbp can have a higher amino acid sequence identity (e.g., have at least about 99%, at least about 95%, at least about 90%, at least about 85%, at least about 80%, or at least about 75% amino acid sequence identity) to the subject fHbp in a fragment (e.g., a variable fragment as defined in the modular architecture) or in a full-length mature protein.
The corresponding fHbp used as a control in the comparison of binding affinity of the subject fHbp may also comprise, in one or more fragments, a fHbp having the same progenitor (α or β) as the corresponding fragment of the subject fHbp.
The subject fHbp can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% amino acid sequence identity to a control fHbp; and differs from the amino acid sequence of the control fHbp by 1 amino acid (aa) to 10 amino acids, e.g., by 1 aa, 2 aa, 3 aa, 4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10 aa from the amino acid sequence of the control fHbp. Thus, for example, a subject fHbp has at most one, at most two, at most three, at most four, up to at most 10 or more modifications (e.g., substitutions, deletions, or insertions) relative to a naturally-occurring and/or non-naturally-occurring (e.g., chimeric) fHbp from which the subject fHbp is derived. The one or more amino acid changes can decrease the affinity of the fHbp for human fH, as compared to an unaltered fHbp. As indicated above, the fHbp from which a subject fHbp is derived comprises a naturally occurring fHbp and a non-naturally occurring fHbp. Non-naturally occurring fHbp can comprise artificially prepared chimeras, such as those well known in the art and those described in PCT application No. WO2009/114485, the disclosure of which is incorporated herein by reference.
Thus, in certain embodiments, a subject fHbp comprises a single amino acid substitution relative to a control fHbp (e.g., where the control fHbp is a naturally occurring fHbp (e.g., fHbp ID1, or a artificially prepared chimera)). In certain embodiments, the subject fHbp comprises a single amino acid substitution (i.e., only one amino acid substitution) relative to a naturally-occurring fHbp (e.g., fHbp ID 6,
By comparing the amino acid sequence of the fH low binding protein (e.g.,
A subject fHbp variant can be derived from (e.g., can comprise one or more amino acid substitutions as compared to) a variant 1fHbp, a variant 2fHbp, or a
Amino acid substitutions that may reduce the affinity of fHbp for fH, as compared to a control fHbp, include amino acid substitutions of contact residues in the fHbp amino acids that bind to fH; surface exposed amino acid substitutions in fHbp amino acids; an amino acid substitution at the interface of the amino-terminal and carboxy-terminal domains in the fHbp amino acid; and amino acid substitutions adjacent to the fH-binding residue in the fHbp amino acid, wherein an amino acid "adjacent" to the fH-binding amino acid is one to ten residues away from the amino terminus or the carboxy terminus of the fH-binding amino acid. In certain embodiments, the amino acid substitution that results in reduced affinity for fH is not an fH contact residue. Some of the contact residues are shown in bold in FIG. 19.
When the fHbp comprises an amino acid substitution relative to naturally occurring or relative to fHbp ID1, the amino acid substitution may be conservative with respect to the amino acid substitution. For example, if R41 is modified to S, resulting in a R41S substitution, and in particular a R41S substitution such that its affinity for human fH is reduced, the disclosure of the present application also includes conservative amino acid substitutions relative to S, and thus also includes amino acid substitutions for R41T.
The present application provides a non-naturally occurring fHbp having an amino acid substitution at
In certain embodiments, amino acid substitutions of one or more of the following residues are specifically excluded: r41, Q38, Q87, Q113, K119, D121, G121, Q126, Q128, R130, D201, E202, E218, a235, E239, and K241. In certain embodiments, for example, when the subject fHbp comprises a single amino acid substitution relative to the control fHbp (e.g., when the control fHbp is a naturally occurring fHbp or fHbp ID 1), the single amino acid substitution can be at position E218 or E239.
Amino acid changes in the subject fHbp include, residues shaded in figure 19 and/or residues in boxes and listed below. In one example of sequence analysis, fragment A (V) of the low binding protein fHbp ID15 was comparedA(ii) a Residues 8-73) V with the same ancestral sequence as in fH high binding protein fHbp ID28A. Thus, in addition to
Other candidate residue positions at which amino acid changes can be introduced are discussed in the examples below. For example, a fHbp of the invention can have amino acid substitutions at one or more positions corresponding to one or more amino acid residues selected from 41, 60, 114, 113, 117, 119, 121, 128, 130, 147, 148, 149, 178, 195, 218, 239, 241, or 247 of fHbp ID1 (e.g., based on the numbering of the mature fHbp). Fhbps of the invention can have amino acid substitutions at one or more positions corresponding to one or more amino acid residues selected from 41, 60, 80, 113, 114, 117, 119, 121, 128, 130, 147, 148, 149, 178, 195, 199, 211, 220, 222, 236, 241, 247, or 248 of fHbp ID1, based on the numbering of the mature fHbp. Fhbps of the invention can have amino acid substitutions at one or more positions corresponding to one or more amino acid residues selected from 87, 109, 115, 118, 126, 138, 197, 201, 202, 203, 209, 217, 225, 235, or 245 of fHbp ID1, based on the numbering of the mature fHbp. When the corresponding fHbp is a
A variant factor H binding protein (fHbp) of the invention may also have amino acid substitutions at one or more positions corresponding to one or more amino acid residues selected from 60, 114, 117, 147, 148, 149, 178, 195, or 247 of
The variant fHbp of the invention may be a variant of fHbp ID1 and may comprise one, two, three, or four of the following substitutions: R41S, R41A, R130A, H119A, E218A, and E239A. As discussed above, the variant fHbp of the present invention may comprise a single amino acid substitution. The variant fHbp of the present invention may also include double amino acid substitutions. For example, a variant fHbp of the invention may comprise two substitutions of R41S, R41A, R130A, H119A, E218A, and E239A.
Variant fhbps of the invention may also have amino acid substitutions at one or more positions corresponding to one or more amino acid residues selected from the group consisting of 80, 211, 218, 220, 222, 236, and 248 of
A variant fHbp of the invention may have amino acid substitutions at one or more positions corresponding to one or more amino acid residues selected from 41, 113, 119, 121 of
A variant fHbp of the invention can have amino acid substitutions at one or more positions corresponding to one or more amino acid residues selected from 113, 121, 199, and 218 of
For example, the corresponding fHbp may be from modular group I, with all variable fragments of the modular group I being of the α lineage, examples of such subject fhbps include R41S mutants of fHbp ID1, 4, 9, and 94.
Chimeric fHbp
As indicated above, a naturally occurring fHbp or a artificially prepared fHbp (e.g., a chimeric fHbp that is artificially prepared) can introduce one or more modifications. Modifications may include modifications in one fragment or one domain, while other fragments and/or domains may be derived from any fHbp (e.g., naturally occurring fhbps of different variant groups).
Having a module architecture V has been describedA、VB、VC、VDAnd VEModifications can be introduced into α lineage V in the fHbp of the fragmentsA(e.g., introduction of R41S in VA of fHbp ID 1), while other fragments of fHbp (e.g., VB、VC、VDAnd VE) Each may be independently derived from any lineage, any variant group, or any fHbp ID. In another example, the V of the subject fHbpA、VCAnd VEFragments may be derived from α lineage (lineage 1), whereas VBAnd VDMay be derived from the β lineage when modified to replace the arginine at
The fHbp of the present invention may be at VAα fragment containing the R41S mutationAα fragments comprise an amino acid sequence having at least about 90%, at least about 92%, at least about 94%, at least about 95%, at least about 96%, at least about 98%, at least about 99%, up to 100% identity to:
Chimeric fhbps of the invention can also be described as having modifications in the N-terminal domain of the fHbp (fHbp N), while the C-terminal domain (fHbp C) can be derived from different fhbps (e.g., different variant groups or different lineages). "fhbp" refers to a contiguous sequence of amino acids that begins at about
The corresponding chimeric fHbp may be any known artificially prepared chimera, such as those described in beerink et al (2008) infec.immun.76: 2568-. Chimeras comprising the modifications have reduced affinity for human fH relative to chimeric fHbp, but retain epitopes that are important for eliciting bactericidal responses, such as the epitopes found in the corresponding chimeric fHbp. Epitopes of fHbp that can be retained in the modified chimeras include epitopes found in the corresponding chimeric fHbp, such as the epitopes described in WO2009/114485, the disclosure of which is incorporated by reference into the present application. For example, the modified chimeric fHbp can comprise an epitope that has an important effect on eliciting a bactericidal response against a strain containing variant 1fHbp (e.g., an epitope in the N-terminal domain, such as an epitope defined by mAb JAR4 and/or JAR 5) and/or a
One characteristic of subject fHbp is that when administered to a host (e.g., a mammal such as a mouse or a human), the subject fHbp can elicit a bactericidal response that is comparable to or slightly higher than the level of bactericidal response elicited by fHbp ID1, or other corresponding controls (e.g., fHbp ID4, 9, 22, 28, 74, or 77). Methods for determining the level of bactericidal response are well known in the art, see the description in the examples section below. For example, the geometric mean of the bactericidal titer of a mouse immunized with subject fHbp is at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 150%, at least about 175%, at least about 200%, or 200% or more of the geometric mean of the bactericidal titer of a mouse immunized with
Subject fHbp can exclude fHbp that elicit a bactericidal response significantly lower than
In many cases, the subject fHbp variants retain and present conformational epitopes that bind bactericidal antibodies that have bactericidal activity against one or more neisseria meningitidis strains. Thus, such fHbp mutants can retain the epitope found in naturally occurring fHbp and have reduced binding to fH as compared to the binding affinity of naturally occurring fHbp to fH. Variants that have minimal or no effect on fHbp configuration, thereby enabling mutant vaccines to elicit bactericidal antibodies, are considered good vaccine candidates. Whether a variant has an effect on the configuration of fHbp can be determined in a variety of ways, including antibody binding as listed in table 9.
fHbp of the invention may have additional features, as described in detail below.
Conjugates
A subject fHbp of the invention can contain one or more additional elements at the N-and/or C-terminus of the polypeptide, such as a polypeptide (e.g., having an amino acid sequence heterologous to the subject fHbp) and/or a carrier molecule. Additional heterologous amino acid sequences can be fused, for example, as a result of expression in a bacterial host cell (e.g., E.coli) to provide an N-terminal methionine or derivative thereof (e.g., pyroglutamate), and/or to provide a chimeric polypeptide having a fusion partner at its N-terminus or C-terminus. Fusion partners of interest include, for example, glutathione-S-transferase (GST), Maltose Binding Protein (MBP), His 6-tag, and the like, as well as leader peptides from other proteins, particularly lipoproteins. Fusion partners may provide additional features, for example, to facilitate isolation, purification, detection, or immunogenicity of the subject fHbp.
Other elements that may be linked to the subject fHbp include carrier molecules (e.g., carrier proteins, such as Keyhole Limpet Hemocyanin (KLH)). Other elements may be linked to the peptide through a linker, e.g., a flexible linker. The carrier comprises an immunomodulator, a molecule that directly or indirectly modifies the immune response. A particular class of immunomodulators includes immunomodulators which stimulate or assist in stimulating an immune response. Examples include antigens and antigenic carriers such as toxins or derivatives thereof, including tetanus toxin. Other vectors are also included that may facilitate administration and/or increase immunogenicity in subjects immunized or treated against neisseria meningitidis. The carrier molecule may also facilitate delivery to a target cell or tissue. The additional moiety may also contribute to immunogenicity or form complexes with components in the vaccine. The carrier molecule may act as a scaffold protein to facilitate display of the membrane surface antigen (e.g., a vesicular vaccine).
In one example, the subject fHbp is modified at its N-and/or C-terminus to introduce fatty acids(e.g., aliphatic carboxylic acid groups). The fatty acid can be covalently linked to the fHbp through a flexible linker. An example of a fatty acid that can be used to modify the terminus of the subject fHbp (e.g., N-terminus, e.g., at the N-terminus) is lauric acid. When covalently linked to other molecules, lauric acid refers to a lauroyl group (e.g., lauroyl sulfate). Lauric acid has twelve carbon atoms and ten methylene groups, and has a molecular formula of CH3-(CH2)10-COOH. Other fatty acids that may be linked to the subject peptide include caprylic (10C), myristic (14C), and palmitic (16C) acids. For details, see Westerink MA et al (1995) Proc. Natl. Acad. Sci. USA 92: 4021-. It is also contemplated that the present application also includes any hydrophobic moiety capable of anchoring a subject fHbp to the bacterial outer membrane, which can be used to couple to the N-and/or C-terminus (e.g., at the N-terminus) of a fHbp of the present invention, where the hydrophobic moiety can be operatively coupled to a peptide via a linker, such as a flexible linker as described herein. For example, the hydrophobic pentapeptide FLLAV (SEQ ID NO:18) is described in Lowell GH et al (1988) J.exp.Med.167: 658-63.
As indicated above, one way of linking fatty acids, as well as other additional elements described above, to fHbp is through a linker (e.g., lauroyl-Gly). Linkers suitable for modifying fHbp of the invention include "flexible linkers". Suitable linkers can be readily selected, and can be of any suitable varying length, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including from 4 amino acids to 10 amino acids, from 5 amino acids to 9 amino acids, from 6 amino acids to 8 amino acids, or from 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4,5, 6, or 7 amino acids.
Examples of flexible linkers include glycine polymers (G)nGlycine-serine polymers (including, for example, (GS)n、GSGGSn(SEQ ID NO:19) and GGGSn(SEQ ID NO:20) where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and others known in the artA flexible connector. Glycine and glycine-serine polymers are preferred because these amino acids are relatively unstructured and therefore can serve as neutral linkages between the components. Glycine polymers are particularly preferred because glycine is able to enter a significantly larger Ramachandran (or phi-psi) space than flat alanine and has lower limitations than residues with longer side chains (see Scheraga, rev. comparative chem.11173-142 (1992)). Exemplary flexible linkers include, but are not limited to, GGSG (SEQ ID NO:21), GGSGG (SEQ ID NO:22), GSGSGSG (SEQ ID NO:23), GSGGG (SEQ ID NO:24), GGGSG (SEQ ID NO:25), GSSSG (SEQ ID NO:26), and the like. One of ordinary skill will recognize that the design of fHbp coupled to any of the elements described above may include a linker that is flexible in whole or in part, such that the linker may include a flexible linker and one or more portions having less flexible structure.
Natural fHbp typically contains an N-terminal cysteine to which a lipid group can be covalently attached. The cysteine residue is typically lipidated in naturally occurring proteins, which may be lipidated in the fHbp of the subject invention. Thus, reference to "cysteine" or "C" at that particular position in the amino acid sequences described herein includes reference to unmodified cysteines and cysteines that are lipidated (e.g., due to post-translational modifications). Thus, the subject fHbp may be lipidated or non-lipidated. Methods for producing lipidated proteins in vitro (see, e.g., Andersson et al (2001) J. immunological Methods 255:135-48) or in vivo are well known in the art. For example, lipidated fHbp has been purified from membrane components of E.coli proteins by detergent extraction methods (Fletcher et al (2004) infection and Immunity 72:2088-100), which may be suitable for the production of lipidated fHbp. Lipidated proteins may be preferred because of their higher immunogenicity compared to soluble proteins (see, e.g., Fletcher et al (2004) Infection and Immunity 72: 2088-.
It is understood that the nucleotide sequence encoding the heterologous fHbp can be modified to optimize the codons used to facilitate expression in a host cell of interest (e.g., e.coli, neisseria meningitidis, human (in the case of DNA-based vaccines), etc.). Methods for producing optimized codon sequences are well known in the art.
Nucleic acids encoding fHbp
The present application provides a nucleic acid encoding a subject fHbp. In certain embodiments, the subject nucleic acid is present in a recombinant expression construct. Also provided are host cells comprising a genetic modification of a subject nucleic acid.
fHbp polypeptides and encoding nucleic acids of the invention may be derived from any suitable neisseria meningitidis strain. As is well known in the art, strains of Neisseria meningitidis are classified as serogroups (capsular group), serotypes (PorB phenotype) and subtypes (PorA phenotype) based on the reactivity of polyclonal (Frasch, C.E. and Hapman,1973, J.Infect.Dis.127:149-154) or monoclonal antibodies that interact with different surface antigens. Capsular grouping is usually based on immunologically detectable differences in capsular polysaccharides, but this detection method is being replaced by PCR encoding specific enzyme genes responsible for the biosynthesis of structurally different capsular polysaccharides. About 12 capsular groups are known (including A, B, C, X, Y, Z, 29-E, and W-135). Strains of capsular group A, B, C, Y and W-135 can cause almost all meningococcal disease. Serotypes are routinely distinguished by antigenic differences in the outer membrane protein known as channel protein b (porb), as determined by monoclonal antibodies. Antibodies known to date can identify about 21 serotypes (Sacchi et al, 1998, clin. diag. lab. immunol.5: 348). The outer membrane protein known as channel protein a (pora) has been distinguished from serosubtypes by determining its antigenic difference by antibodies. Serotyping and serosubtyping are being replaced by PCR and/or DNA sequencing, which identifies genes encoding the PorB and PorA variable regions, respectively, that are involved in mAb reactivity (e.g., Sacchi, Lemos et al, supra; Urwin et al, 1998, epidem. andInfect.120: 257).
Although any capsular group of a neisseria meningitidis strain may be used, it is particularly preferred that a neisseria meningitidis strain of capsular group B is used as the source of the nucleic acid encoding fHbp, and derivatives of domains thereof.
Nucleic acids encoding fHbp polypeptides, which are well known in the art, can be used to construct subject fhbps included herein. Different fhbps and their sequences are available on the neissemia.org and pubmlst.org/neissemia/fHbp websites. Examples of fHbp polypeptides can also be found in, for example, US patent application No. 61/174,424, PCT application No. PCT/US09/36577, WO 2004/048404; masignani et al (2003) J Exp Med197: 789-; fletcher et al (2004) feed Immun72: 2088-; welsch et al.J Immunol 2004172: 5606-; and WO 99/57280. Nucleic acids (and amino acid sequences) of fHbp variants and subvariants are also found in GenBank under accession numbers: NC-003112, GeneID:904318(NCBI Ref. NP-274866), fHbp ID1 from Neisseria
To identify the relevant amino acid sequences contemplated for use in fHbp of the subject invention, it should be noted that the immature fHbp includes a leader sequence of about 19 residues. Furthermore, when an amino acid sequence is provided, one of ordinary skill in the art will readily recognize nucleic acid sequences that encode and express a polypeptide having such an amino acid sequence.
In addition to the specific amino acid sequences and nucleic acid sequences provided herein, the invention also encompasses polypeptides and nucleic acid sequences that are at least 80%, at least 85%, at least 90%, or at least 95% identical to these examples of polypeptide and nucleic acid sequences. The term "identical" or percent "identity," in the context of two or more polynucleotide sequences, or two or more amino acid sequences, refers to within a specified region, e.g., VEOr at least the length in the reference amino acid or
In making sequence comparisons, one sequence is typically used as a control sequence (e.g., a naturally occurring fHbp polypeptide sequence or fragment thereof) to which test sequences are compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer program, sequence coordinates are set, if necessary, and the parameters of the sequence algorithm program are set. The sequence comparison algorithm will then calculate the percent sequence identity of the test sequence to the control sequence based on the set program parameters.
Examples of algorithms suitable for determining percent sequence identity are the BLAST and BLAST 2.0 algorithms, described in Altschul et al (1990) J.mol.biol.215: 403-. Software for performing BLAST analysis is publicly available from the national center for Biotechnology information (www.ncbi.nlm.nih.gov). Further exemplary algorithms include ClustalW (Higgins D., et al (1994) Nucleic Acids Res 22: 4673-.
Certain residues differ in position by conserved amino acids. Conservative amino acid substitutions refer to the interchange of residues with 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 with aromatic side chains is phenylalanine, tyrosine and tryptophan; a group of amino acids having acidic side chains is aspartic acid and glutamic acid; a group of amino acids having a base side chain is lysine, arginine and histidine; and a group of amino acids having sulfur-containing side chains are cysteine and methionine.
Sequence identity between two nucleic acids can also be described using hybridization between two molecules under stringent conditions. Hybridization conditions are selected from the following standard methods in the art (see, e.g., Sambrook, et al, molecular cloning: A Laboratory Manual, 2 nd edition, (1989) Cold Spring Harbor, N.Y.). An example of stringent hybridization conditions is hybridization at 50 ℃ or more and 0.1 XSSC (15mM sodium chloride/1.5 mM sodium citrate). Another example of stringent hybridization conditions is overnight incubation at 42 ℃ in the following solution: 50% formamide, 5 XSSC (150mM NaCl, 15mM trisodium citrate), 50mM sodium phosphate (pH7.6), 5 XDenhardt's solution, 10% dextran sulfate, and 20mg/ml denatured sheared salmon sperm DNA, followed by washing the filter using 0.1 XSSC at about 65 ℃. Stringent hybridization conditions are hybridization conditions that are at least as stringent as the representative conditions described above, wherein conditions are considered to be at least stringent when the stringency is at least about 80%, typically at least 90%, of the stringency conditions specified above.
Production method
fHbp of the invention can be produced by any suitable method, including recombinant and non-recombinant methods (e.g., chemical synthesis). When recombinant techniques are used to produce the subject fHbp, the method may involve any suitable construct and any suitable host cell, either prokaryotic or eukaryotic, typically bacterial or yeast host cells, more typically bacterial cells. Methods for introducing genetic material into a host cell include, for example, transformation, electroporation, conjugation, and the calcium phosphate method, among others. The method of transfer may be selected such that the fHbp encoding nucleic acid to be introduced is stably expressed. The fHbp encoding nucleic acid may be provided as a heritable episomal element (e.g., a plasmid) or integrated into the genome.
Suitable vectors for transferring fHbp encoding nucleic acids may vary in composition. The integrating vector may be conditionally replicable, or may be a suicide plasmid, phage, or the like. The construct may include a variety of elements, including, for example, a promoter, a selectable genetic marker (e.g., a gene resistant to an antibiotic (e.g., kanamycin, erythromycin, chloramphenicol, or gentamicin)), an origin of replication (e.g., initiation of replication in a host cell, e.g., a bacterial host cell), and the like. The choice of vector depends on a variety of factors, such as the type of cell desired for propagation and the purpose of propagation. Certain vectors are useful for amplifying and preparing large quantities of a target DNA sequence. Other vectors are suitable for expression in cultured cells. Still other vectors are suitable for transfer and expression in whole animal cells. Selection of an appropriate vector is well known in the art. Many such vectors are commercially available.
In one example, the vector is an expression vector based on an episomal plasmid that contains selectable drug resistance markers and elements and can provide autonomous replication in different host cells (e.g., E.coli and N.meningitidis). An example of such a "shuttle vector" is the plasmid pFP10 (Patotto et al (2000) Gene 244: 13-19).
Constructs may be prepared, for example, by inserting the polynucleotide of interest into the backbone of the construct, typically by ligation using a DNA ligase to the cleaved restriction enzyme sites in the vector. Alternatively, the target nucleotide sequence may be inserted by homologous recombination or site-specific recombination. Homologous recombination is typically accomplished by ligating the homologous regions to the vector flanking the nucleotide sequence of interest, while site-specific recombination is accomplished by employing sequences that promote site-specific recombination (e.g., cre-lox, att sites, etc.). Nucleic acids containing this sequence can be added, for example, by the following methods: ligation of oligonucleotides, or polymerase chain reaction using primers comprising a region of homology and a portion of the nucleotide sequence of interest.
The vector may remain extrachromosomal in the host cell, or may integrate into the host cell genome. Numerous publications known to those skilled in the art describe in detail vectors, including, for example, Short Protocols in molecular biology, (1999) f. The vector may express a nucleic acid encoding the subject fHbp, may amplify the subject nucleic acid, or both.
Exemplary vectors that can be used include, but are not limited to, vectors derived from recombinant phage DNA, plasmid DNA, or cosmid DNA. For example, plasmid vectors such as pBR322,
For expression of subject fHbp, an expression cassette can be employed. Accordingly, the present invention provides recombinant expression vectors comprising a subject nucleic acid. The expression vectors provide transcriptional and translational regulatory sequences, and may provide inducible or constitutive expression, with the coding regions being operably linked under the transcriptional control of a transcriptional initiation region, and transcriptional and translational termination regions. These control regions may be native to the fHbp from which the subject fHbp is derived, or derived from an external source. Typically, the transcriptional and translational regulatory sequences include, but are not limited to, promoter sequences, ribosome binding sites, transcriptional initiation and termination sequences, translational initiation and termination sequences, and enhancer or activator sequences. The promoter may be constitutive or inducible, and may be a strong constitutive promoter (e.g., T7, etc.).
Expression vectors typically have convenient restriction sites located near the promoter sequence for insertion of the nucleic acid sequence encoding the protein of interest. An operable selectable marker may be present in the expression host to facilitate selection of cells comprising the vector. Furthermore, the expression construct may comprise further elements. For example, the expression vector may have one or two replication systems, thereby enabling it to be maintained in the organism used for expression, e.g., mammalian or insect cells, and prokaryotic hosts for cloning and amplification. In addition, the expression construct may comprise a selectable marker gene to allow selection of transformed host cells. Selection genes are well known in the art and vary with the host cell employed.
It is noted that fhbps of the invention may comprise other elements, such as detectable labels, e.g., radiolabels, fluorescent labels, biotin labels, and immunologically detectable labels (e.g., HA tags, poly-histidine tags), among others. Additional elements of fHbp can be provided to facilitate separation (e.g., biotin tags, immunologically detectable tags) by a variety of methods (e.g., affinity capture, etc.). The subject fHbp may optionally be immobilised to the support by covalent or non-covalent attachment.
Isolation and purification of fHbp can be accomplished according to methods known in the art. For example, fHbp can be isolated from cell lysates or synthesis reaction mixtures that are genetically modified to express fHbp by immunoaffinity purification, which typically involves: contacting the sample with an anti-fHbp antibody (e.g., an anti-fHbp mAb such as JAR 5mAb or other suitable JAR mAb known in the art), washing to remove non-specifically bound material, and eluting specifically bound fHbp. The isolated fHbp can be further purified by dialysis and other methods commonly used for protein purification. In one example, fHbp can be isolated using metal chelate chromatography.
Host cell
A variety of suitable host cells can be used to produce fHbp. In general, the fHbp of the invention may be expressed in prokaryotic or eukaryotic cells, typically bacteria, more typically e.coli or neisseria (e.g. neisseria meningitidis), according to conventional techniques. Accordingly, the present invention further provides a genetically modified host cell comprising a nucleic acid encoding a subject fHbp. The host cell used to produce (including large scale production of) the subject fHbp may be selected from any of a variety of available host cells. Examples of host cells for expression include cells of prokaryotic or eukaryotic unicellular organisms, such as bacteria (e.g., E.coli strains), yeast (e.g., Saccharomyces cerevisiae, Pichia pastoris, and the like), and may include host cells derived from higher organisms, such as insects, vertebrates, and in particular mammals (e.g., CHO, HEK, and the like). In general, in the production of the subject fHbp, bacterial host cells and yeast are particularly preferred.
The subject fHbp can be prepared in a substantially purified or substantially isolated form (i.e., substantially isolated from other neisseria or host cell polypeptides) or in a substantially isolated form. The subject fHbp can be present in a composition that is enriched compared to other components that may be present in the composition (e.g., other polypeptides or other host cell components). Purified subject fHbp can be provided such that the polypeptide is present in a composition that is substantially free of other expressed polypeptides, e.g., less than 90%, typically less than 60%, and more typically less than 50% of the composition consists of other expressed polypeptides.
Host cell for vesicle production
When the subject fHbp is provided in a membrane vesicle (discussed in detail below), the neisserial host cell will be genetically modified to express the subject fHbp. In the methods disclosed herein, any of a variety of neisserial strains that can be modified to produce the subject fHbp, and optionally, can produce or can be modified to produce other antigens of interest, such as PorA, can be used.
Methods and vectors for genetic modification of neisserial strains and expression of polypeptides of interest are well known in the art. See WO 02/09746 and O' Dwyer et al (2004) InfectImmun72:6511-80 for examples of vectors and methods. Strong promoters, in particular strong constitutive promoters, are particularly preferred. Examples of promoters include the promoters of porA, porB, lbpB, tbpB, p110, hpuAB, lgtF, opa, p110, lst, hpuAB, and rmp.
For use in membrane vesicle production, pathogenic neisseria strains or strains derived from neisseria, in particular strains pathogenic to humans or strains pathogenic or commensal to humans, are preferred. Examples of neisseria species include neisseria meningitidis, neisseria aureofaciens, neisseria gonorrhoeae, neisseria lactis, neisseria polysaccharea, neisseria griseus, neisseria mucosae, neisseria flavescens, neisseria desiccation, neisseria elongata, and the like.
It is particularly preferred to use a neisseria meningitidis strain genetically modified to express the subject fHbp and produce vesicles. The strains used for vesicle production may be selected according to a number of different target characteristics. For example, the strains may be selected according to: the type of targeted PorA ("serosubtype", as described above), capsular group, serotype, etc.; reduced production of capsular polysaccharides, and the like. For example, the production strain may produce any PorA polypeptide of interest, and may express one or more PorA polypeptides (native or genetically engineered). Examples of such strains include, but are not limited to, strains producing a strain capable of conferring a serosubtype P1.7, 16; p1.19, 15; p1.7, 1; p1.5, 2; p1.22a, 14; p1.14; p1.5, 10; p1.7, 4; strains of PorA polypeptide of P1.12, 13; and strains that produce such PorA polypeptide variants that may or may not retain reactivity with conventional serological reagents used in serosubtyping. Also preferred are PorA polypeptides identified according to the porA Variable Region (VR) (see, e.g., Russell et al (2004) embedding infection Dis10: 674-. A large number of different VR types have been identified, which can be classified as VR1 and VR2 family "prototypes". A network access database describing this term and its relationship to previous typing schemes is found in neissemia. For some comparisons of VR1 and VR 2-type PorA, please see Russell et al (2004) Emerging Infect Dis10: 674-678.
Alternatively or additionally, the production strain may be a capsule deficient strain. Capsular deficient strains may provide a vesicle vaccine that may reduce the risk of eliciting a significant autoantibody response in a subject to whom the vaccine is administered (e.g., due to the production of antibodies that cross-react with sialic acid on the host cell surface). As used herein, "capsular defect" or "lack of capsular polysaccharide" means that the level of capsular polysaccharide on the surface of the bacterium is lower than that of the naturally occurring strain, or, when the strain is genetically modified, that the level of capsular polysaccharide on the surface of the bacterium is lower than that of the parent strain from which the capsular deficient strain was derived. Capsular deficient strains include strains that have at least a 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, 90% or more reduction in surface capsular polysaccharide production, as well as strains that have no detectable capsular polysaccharide on the bacterial surface (e.g., by whole cell enzyme-linked immunosorbent assay (ELISA) using anti-capsular polysaccharide antibodies).
Capsule deficient strains include strains that are deficient in capsule due to naturally occurring or recombinantly produced genetic modifications. Naturally occurring strains deficient in capsule (see, e.g., Dolan-Livenwood et al (2003) J. Infect. Dis.187:1616-28), and methods for identifying and/or producing strains deficient in capsule (see, e.g., Fisseha et al (2005) Infect. Immun.73: 4070-.
Modifying the neisseria host cell to reduce capsular polysaccharide production may comprise modifying one or more genes involved in capsular synthesis, which modification may reduce the level of capsular polysaccharide compared to the parent cell prior to modification. Such genetic modifications include altering the nucleotide and/or amino acid sequence of one or more capsule biosynthesis genes such that the strain is capsular-deficient (e.g., due to one or more insertions, deletions, substitutions, etc. in one or more capsule biosynthesis genes). The capsule deficient strain may lack one or more capsule genes, or the genes may be non-functional.
Particularly preferred are strains which are defective in the biosynthesis of sialic acid. Such strains can produce vesicles that can reduce the risk of eliciting anti-sialic acid antibodies that cross-react with human sialic acid antigens, and can also improve production safety. Strains with sialic acid biosynthesis defects (either as a result of naturally occurring modifications or engineered modifications) can be defective in any of a number of different genes in the sialic acid biosynthesis pathway. Particularly preferred are strains which are deficient in the gene product encoded by the N-acetylglucosamine-6-phosphate 2-epimerase gene (designated synX AAF40537.1 or siaA AAA20475), particularly preferred are strains in which this gene is inactivated. For example, in one embodiment, a capsular deficient strain is generated by disrupting the production of a functional synX gene product (see, e.g., Swartley et al (1994) JBacteriol.176: 1530-4).
Non-recombinant techniques can also be used to obtain capsular deficient strains from naturally occurring strains, for example by selection of strains with reduced capsular polysaccharide using bactericidal anti-capsular antibodies.
When the invention involves the use of two or more strains (e.g., to produce an antigenic composition comprising vesicles from different strains with subject fHbp), strains may be selected that differ in one or more of the characteristics of the strains, e.g., such that vesicles are provided with different subjects fHbp, PorA, etc.
Preparation of vesicles
The antigenic compositions of the invention generally include vesicles prepared from neisserial cells expressing the subject fHbp. "vesicles" as used herein include outer membrane vesicles as well as microvesicles (also known as blebs).
The antigenic composition may comprise Outer Membrane Vesicles (OMVs) prepared from the outer membrane of a cultured strain of neisseria meningitidis genetically modified to express the subject fHbp. OMVs are obtainable by: culturing Neisseria meningitidis in a broth or solid medium, preferably by separating bacterial cells from the medium (e.g., precipitating the cells by filtration or low speed centrifugation, etc.), lysing the cells (e.g., by adding detergents, osmotic shock, sonication, cavitation, homogenization, etc.), and separating the outer membrane fraction from cytoplasmic molecules (e.g., by filtration; or by differential precipitation or aggregation of outer membrane and/or outer membrane vesicles; or by affinity separation methods using ligands that specifically recognize outer membrane molecules; or by high speed centrifugation, etc.); OMVs can be prepared using the outer membrane component.
The antigenic composition can comprise Microvesicles (MVs) (or "blebs") comprising a subject fHbp, wherein a neisseria meningitidis strain genetically modified to express the subject fHbp releases the MVs or blebs during culture. For example, MV can be obtained by: culturing neisseria meningitidis in a broth medium, separating intact cells from the broth medium (e.g., by filtration or low speed centrifugation, thereby precipitating only cells, but not smaller blebs, etc.), and then collecting MV in the cell-free medium (e.g., by filtration, differential precipitation or aggregation of MV, or by high speed centrifugation, etc.). The strain used to produce MV may generally be selected based on the number of blebs produced in the culture medium (e.g., a reasonable number of bacteria may be cultured to produce blebs suitable for isolation and administration in the methods described herein). Exemplary strains that produce high levels of blebs are described in PCT publication WO 01/34642. In addition to cell vesicle production, strains for producing MV can also be selected based on NspA production, with strains that produce higher NspA levels (examples of Neisseria meningitidis with different NspA production levels are described, for example, in Moe et al (1999 infection. Immun.67: 5664.) other strains of interest for producing cell vesicles include strains with an inactive GNA33 gene that encodes lipoproteins required for cell isolation, membrane structure and toxicity (see, for example, Adu-Bobie et al (2004) infection Immun.72: 1914-.
The antigenic compositions of the invention may contain vesicles from one strain, or 2, 3, 4,5 or more strains, which may be homologous or heterologous, usually heterologous, to each other. For example, the strain may be homologous or heterologous with respect to the fHbp from which PorA and/or subject fHbp are derived. Vesicles can be prepared using strains that express more than one subject fHbp (e.g., 1, 2, 3, or more subject fhbps), which can consist of amino acid sequences of different variants (v.1, v.2, or v.3) or subvariants (e.g., subvariants of v.1, v.2, or v.3) fhbps.
The antigenic composition can comprise a mixture of OMVs and MVs that provide fHbp in the same or different subjects, wherein the subject fHbp can optionally provide epitopes from different combinations of fHbp variants and/or subvariants, and wherein the OMVs and/or MVs can be from the same or different strains. Vesicles from different strains may be administered as a mixture or sequentially.
Where desired (e.g., the strain used to produce the vesicles is associated with endotoxin or a particularly high level of endotoxin), the vesicles may optionally be treated to reduce endotoxin, e.g., to reduce toxicity after administration. Endotoxin can be reduced by extraction with a suitable detergent (e.g., BRIJ-96, sodium deoxycholate, sodium lauroyl sarcosinate, Empigen BB, Triton X-100, Tween 20 (polyoxyethylene sorbitan monolaurate),
Vesicles of the antigenic composition may be prepared without the use of detergents, e.g. without the use of deoxycholate. While detergent treatment can be used to remove endotoxin activity, native fHbp lipoproteins and/or subject fHbp (including lipidated fHbp) may be removed by extraction steps during vesicle product preparation. Thus, it is particularly desirable to reduce endotoxin activity using techniques that do not require detergents. In one approach, strains that produce less endotoxin (lipopolysaccharide, LPS) are used, thereby avoiding removal of endotoxin from the final preparation prior to use in humans. For example, vesicles may be prepared using neisserial mutants in which lipooligosaccharides or other antigens that may be harmful in the vaccine (e.g., Rmp) are reduced or eliminated.
Vesicles can be prepared using strains of neisseria meningitidis that include genetic modifications such that the toxic activity of lipid a is reduced or undetectable. For example, the strain may be genetically modified for lipid A biosynthesis (Steeghs et al (1999) infusion Immun67: 4988-93; van der Ley et al (2001) infusion Immun69: 5981-90; Steeghs et al (2004) J Endotoxin Res10: 113-9; Fissha et al (2005) infusion Immun73: 4070). The immunogenic composition may be detoxified by modifying LPS, for example by downregulating and/or inactivating the enzyme encoding lpxL1 or lpxL2, respectively. Pentaacylated lipid a was produced in the lpxL1 mutant, suggesting that the enzyme encoded by lpxL1 adds C12 to the N-linked 3-OH C14 at the 2' position of glcnii. In the lpxL2 mutant, the predominant lipid a species was the tetraacylate, suggesting that the enzyme encoded by lpxL2 adds an additional C12 at the 2-position of GlcN I, i.e., to the N-linked 3-OH C14. Mutations result in reduced (or no) expression of these genes (or reduced or no activity of the products of these genes) and altered lipid A toxicity (van der Ley et al (2001) infection Immun69: 5981-90). Tetra-acylated (lpxL2 mutant) and pentaacylated (lpxL1 mutant) lipid a were less toxic than wild-type lipid a. Mutations in the lipid A4' -kinase encoding gene (lpxK) can also reduce the toxic activity of lipid a. For vesicle (e.g., MV or OMV) production, particularly preferred is neisseria meningitidis that is genetically modified such that the protein encoded by functional LpxL1 is reduced or undetectable, e.g., a neisseria (e.g., a neisseria meningitidis strain) is genetically modified such that the activity of the LpxL1 gene product is reduced or inactive. For example, a neisseria species bacterium can be genetically modified to knock out the lpxL1 gene, e.g., to disrupt the lpxL1 gene. See, for example, U.S. patent publication No. 2009/0035328. The neisseria species bacterium may be genetically modified such that the lpxL2 gene product is reduced or inactive. The neisseria species bacterium may be genetically modified such that the activity of the lpxL1 gene and lpxL2 gene products is reduced or inactive. Such vesicles have reduced toxicity compared to wild-type n.meningitidis strains that are capable of producing LPS, but still retain the immunogenicity of fHbp.
It is also possible to alter the toxicity of LPS by introducing mutations in the gene/locus involved in polymyxin B resistance (which is associated with the addition of aminoarabinose to the 4' phosphate group of lipid a). These genes/loci can be either pmrE, which encodes UDP-glucose dehydrogenase, or a region of the antimicrobial peptide resistance gene that is common to many enterobacterins that are involved in amino arabinose synthesis and transfer. The pmrF gene present in this region encodes dolichol-phosphate mannosyltransferase (Gunn J.S., Kheng, B.L., Krueger J., Kim K., Guo L., Hackett M., Miller S.I.1998.mol. Microbiol.27: 1171-1182).
Mutations in the PhoP-PhoQ regulatory system, which is a phosphorylated two-component regulatory system (e.g., PhoP constitutive phenotype, PhoPc), or low Mg + + environment or culture conditions that activate the PhoP-PhoQ regulatory system, can result in the addition of aminoarabinose to the 4' phosphate and the replacement of nutmeg with 2-hydroxynutmeg (nutmeg hydroxylation). The modified lipid A has reduced ability to stimulate E-selectin expression by human endothelial cells and TNF secretion by human monocytes.
Polymyxin B resistant strains are also suitable because of the reduced LPS toxicity of these strains (see, e.g., van der Ley et al (1994) In: Proceedings of the national nutritional Neisseria conference, the Guildhall, Winchester, England). Alternatively, synthetic peptides that mimic the polymyxin B binding activity may be added to the antigenic composition to reduce LPS toxic activity (see, e.g., Rustici et al (1993) Science 259: 361-365; Porro et al (1998) Prog Clin Biol Res.397: 315-25).
Endotoxin can also be reduced by selection of culture conditions. For example, the strain is cultured in a growth medium containing 0.1mg to 100mg of aminoarabinose per liter to reduce lipid toxicity (see, e.g., WO 02/097646).
Compositions and formulations
As used herein, a "composition," "antigenic composition," or "immunogenic composition" are generally short for compositions comprising a subject fHbp of the present invention, which may optionally be conjugated to further enhance immunogenicity. Specifically included in the invention are compositions for eliciting antibodies in humans, such as antibodies against neisseria meningitidis, such as bactericidal antibodies against neisseria meningitidis. The antigenic composition may comprise 1, 2 or more different subject fhbps. When more than one type of fHbp is present, each subject fHbp may provide epitopes from different combinations of fHbp variants and/or subvariants.
The antigenic composition contains an immunologically effective amount of the subject fHbp and may further comprise other compatible components, if desired. The compositions of the invention may contain fHbp as a fH low binding protein. fH low binding proteins in the subject compositions include any fHbp described above, such as non-naturally occurring or naturally occurring fHbp (e.g.,
Immunogenic compositions of the invention include, but are not limited to, compositions comprising:
1) a non-naturally occurring fHbp (e.g., a non-naturally occurring fHbp that has a lower affinity for human fH than fHbp ID 1); and
2) fHbp (e.g., non-naturally occurring fHbp, e.g., non-naturally occurring fHbp with lower affinity for fH than fHbp ID 1) and NspA;
wherein the fHbp and/or NspA may be provided as recombinant proteins and/or in a vesicle composition (e.g., OMV). It should be noted that when the composition includes NspA and fHbp, the bactericidal activity of the antibody elicited by administration of the composition is the result of the co-action of the antibodies against one or both antigens. Examples of immunogenic compositions provided by the invention include:
a) an immunogenic composition comprising a non-naturally occurring fHbp variant described above (e.g., a non-naturally occurring fHbp that elicits a bactericidal antibody response against at least one neisseria meningitidis strain);
b) an immunogenic composition comprising a non-naturally occurring fHbp variant described above (e.g., a non-naturally occurring fHbp having a lower affinity for human fH than fHbp ID 1); and a recombinant NspA protein;
c) an immunogenic composition comprising an isolated fHbp having at least 85% amino acid sequence identity to
d) an immunogenic composition comprising an isolated fHbp, and a recombinant NspA protein; the isolated fHbp has at least 85% amino acid sequence identity to
e) an immunogenic composition comprising a native OMV from a genetically modified neisseria host cell that is genetically modified with a nucleic acid encoding a non-naturally occurring fHbp variant as described above (e.g., a non-naturally occurring fHbp that has lower affinity for human fH than fHbp ID 1), such that the genetically modified host cell produces the encoded non-naturally occurring fHbp, wherein the OMV comprises the encoded non-naturally occurring fHbp; and
f) an immunogenic composition comprising a native OMV from a genetically modified neisseria host cell that is genetically modified with a nucleic acid encoding a non-naturally occurring fHbp variant as described above, (e.g., a non-naturally occurring fHbp that has a lower affinity for human fH than fHbp ID 1), such that the genetically modified host cell produces the encoded non-naturally occurring fHbp, wherein the OMV comprises the encoded non-naturally occurring fHbp; and the neisserial host cell also produces high levels of NspA such that the OMV also contains NspA. For example, the neisserial host cell may be genetically modified such that NspA expression is increased.
By "immunologically effective amount" is meant that the amount, when administered to an individual, whether as a single dose or as part of sequential administration of the same or different immunogenic compositions, is effective to elicit an effective antibody response useful for treating or preventing a condition or disease caused by, for example, infection by a neisseria species, particularly neisseria meningitidis, and more particularly neisseria meningitidis group B. The amount varies depending on the following factors: the health and physical condition of the individual to be treated, age, the ability of the individual's immune system to produce antibodies, the degree of protection targeted, vaccine formulation, the attending physician's assessment of the medical condition, and other relevant factors. It is contemplated that the amount may be in a relatively wide range, which can be determined by routine experimentation.
The amino acid sequence of an NspA polypeptide is well known in the art. See, for example, WO 96/29412; and Martin et. (1997) j.exp.med.185: 1173; GenBank accession No. U52066; and GenBank accession number AAD 53286. An "NspA polypeptide" can comprise an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to a contiguous chain of about 75 amino acids to about 100 amino acids, about 100 amino acids to about 150 amino acids, or about 150 amino acids to about 174 amino acids in the amino acid sequence set forth in fig. 44. An "NspA polypeptide" can comprise an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to a contiguous chain of about 75 amino acids to about 100 amino acids, or about 100 amino acids to about 155 amino acids, of 20 to 174 amino acids in the amino acid sequence set forth in fig. 44. In certain instances, the NspA polypeptide lacks a signal sequence; in other cases (e.g., expressed in a host cell), the NspA polypeptide includes a signal sequence.
The dosage regimen may be a single dose regimen or a multiple dose regimen (e.g., including booster doses) that administers the antigenic composition in unit dosage forms at different times. The term "unit dosage form" as used herein refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of an antigenic composition of the invention sufficient to produce the desired effect, said composition being provided in association with a pharmaceutically acceptable excipient (e.g., a pharmaceutically acceptable diluent, carrier or solvent). The antigenic composition may be administered in combination with other immune modulators.
The antigenic composition is provided in a pharmaceutically acceptable excipient, which may be a solution, such as a sterile aqueous solution, typically a physiological saline solution, or a powder. If necessary, the adjuvant is substantially inert.
In certain embodiments, the subject immunogenic composition comprises a subject fHbp present in a vesicle. In certain embodiments, the subject immunogenic composition comprises a subject fHbp present in MV. In certain embodiments, the subject immunogenic composition comprises a subject fHbp present in an OMV. In certain embodiments, the subject immunogenic composition comprises a mixture of MV and OMV comprising the subject fHbp. Vesicles, such as MV and OMV, for example, have been described above.
The antigenic composition may further comprise an adjuvant. Examples of known suitable adjuvants that may be used in humans include, but are not necessarily limited to: alum, aluminum phosphate, aluminum hydroxide, MF59 (4.3% w/v squalene, 0.5% w/v tween 80TM, 0.5% w/v span 85), CpG containing nucleic acids (where cytosine is not methylated), QS21, MPL, 3DMPL, Aquilla extract, ISCOMS, LT/CT mutants, poly (D, L-lactide-co-glycolide) (PLG) microparticles, Quil a, interleukins, and the like. For the experimental animals, freund's adjuvant, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-N-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as N-MDP), N-acetyl-muramyl-L-alanyl-D-isoglutamine-L-alanine-2- (1' -2' -di-palmitoyl-sn-glycero-3-hydroxyphosphoryloxy) -ethylamine (CGP19835A, referred to as MTP-PE), and RIBI containing three components extracted from bacteria in a 2% squalene/
Further exemplary adjuvants that can enhance the efficacy of the composition include, but are not limited to: (1) oil-in-water emulsions (with or without other specific immunostimulants, such as muramyl peptides (see below) or bacterial cell wall components), for example (a) MF59TMContaining 5% squalene, 0.5% tween 80 and 0.5% span 85 (optionally containing MTP-PE) (WO 90/14837; Chapter 10in Vaccine design: the subBunit and adjuvant aproach, eds&Newman, Plenum Press 1995), using a microfluidizer to make submicron particles; (b) SAF containing 10% squalene, 0.4% Tween 80, 5% pluronic block polymer L121, and thr-MDP, microfluidized into submicron emulsion, or vortexed to produce emulsion with larger particle size, and (c) RIBITMAdjuvant System (RAS) (Ribi Immunochem, Hamilton, Mont.) containing 2% squalene, 0.2% tween 80, and one or more bacterial cell wall components, such as monophosphoryl lipid a (MPL), Trehalose Dimycolate (TDM), and Cell Wall Skeleton (CWS), preferably MPL + CWS (Detox @)TM) (ii) a (2) Saponin adjuvants, e.g.To use QS21 or StimulonTM(Cambridge Bioscience, Worcester, Mass.) or particles produced therefrom, such as ISCOMs (immune stimulating complexes) which may lack additional detergents, such as WO 00/07621; (3) complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (4) cytokines such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12(WO99/44636), etc.), interferons (e.g., gamma interferon), macrophage colony stimulating factor (M-CSF), Tumor Necrosis Factor (TNF), etc.; (5) monophosphoryl lipid ｃA (MPL) or 3-O-deacylated MPL (3dMPL), see for example GB-2220221, EP- ｃA-0689454, optionally substantially free of alum when used in combination with pneumococcal saccharides, e.g. WO 00/56358; (6)3dMPL in combination with, for example, QS21 and/or an oil-in-water emulsion, such as EP- ｃA-0835318, EP- ｃA-0735898, EP- ｃA-0761231; (7) oligonucleotides containing CpG motifs (Krieg Vaccine2000,19, 618-622; Krieg Curr opin Mol Ther 20013: 15-24; Roman et al, Nat.
The antigen composition may comprise other ingredients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium, carbonate, and the like. The compositions may contain pharmaceutically acceptable auxiliary substances as required to simulate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like.
The concentration of subject fHbp in the formulation can vary widely (e.g., from less than about 0.1%, usually or at least about 2% up to 20% to 50% or higher by weight), and is selected primarily according to the particular mode of administration chosen and the needs of the patient, according to fluid volume, viscosity, and patient-based factors.
Formulations comprising fHbp may be provided in the form of solutions, suspensions, tablets, pills, capsules, powders, gels, creams, lotions, ointments, aerosols, and the like. It is known that oral administration requires protection of the composition from digestion. The compositions are generally protected by formulating the compositions to be resistant to acid and enzymatic hydrolysis, or by coating the compositions with a suitable resistance carrier. Methods of protecting against digestion are well known in the art.
Formulations comprising fHbp may also be provided such that the serum half-life of fHbp is increased following administration. For example, when fHbp alone is formulated as an injection, the fHbp can be formulated as a liposome formulation, as a colloid, or other conventional formulation that can extend serum half-life. Liposomes can be prepared by a variety of methods, see, e.g., Szoka et al, ann.rev.biophysis.bioeng.9: 467(1980), U.S. patent nos. 4,235,871, 4,501,728, and 4,837,028. The formulation may also be formulated as a controlled or sustained release formulation.
Immunization
The present invention provides a method of inducing an immune response in a mammalian host against at least one strain of neisseria. The methods generally involve administering to a subject in need thereof an effective amount of an immunogenic composition.
Antigenic compositions comprising fHbp are typically administered to humans at risk of acquiring neisserial disease to prevent or at least partially arrest the development of the disease and its complications. An amount sufficient to achieve this purpose is defined as a "therapeutically effective amount". A therapeutically effective amount will depend, for example, on the antigenic composition, the mode of administration, the weight and general health of the patient, and the judgment of the prescribing physician. Whether the antigenic composition is administered in a single dose or in multiple doses depends on the dose and frequency required and tolerated by the patient, and the route of administration.
The antigenic composition comprising fHbp is typically administered to a host in an amount effective to elicit an immune response, particularly a humoral immune response, such as a bactericidal antibody response. As noted above, the amount of immunization can vary, and is generally used in an amount ranging from about 1. mu.g to 100. mu.g per 70kg of patient, more usually 5. mu.g to 50. mu.g per 70 kg. Oral, nasal, or topical administration may employ substantially higher doses (e.g., 10mg to 100mg or higher). The same or different antigenic compositions comprising fHbp may be used for booster immunizations following initial administration. Typically, vaccination is at least one boost, more typically two boosts.
In general, immunization may be administered by any suitable route, including orally, nasally, nasopharyngeally, parenterally, enterally, gastrically, topically, subcutaneously, intramuscularly, in the form of tablets, solids, powders, liquids, aerosols, topically or systemically, with or without the addition of adjuvants. For a more detailed description of the actual methods of preparing parenterally administrable compositions known or apparent to those skilled in the art, see, e.g., Remington's Pharmaceutical Science, 15 th edition, Mack Publishing Company, Easton, Pa. (1980).
The immune response against fHbp can be assessed by known methods (e.g., individual sera collected before and after the first immunization, respectively, demonstrating an alteration in the immune status of the individual by, for example, immunoprecipitation assays, or ELISA, or bactericidal assays, or Western blotting, or flow cytometry assays, etc.).
The antigenic composition may be administered to a human subject who has not been immunised with neisseria meningitidis (immunogically nasal). In a particular embodiment, the subject is a child about 5 years of age or less, preferably a child about two years of age or less, and the antigenic composition is administered at one or more of the following times: two weeks after birth, 1, 2, 3, 4,5, 6, 7, 8, 9, 10 or 11 months, or 1 year or 15, 18 or 21 months, or at the age of 2, 3, 4 or 5 years.
It is generally desirable to perform a first immunization prior to the first signs of disease symptoms, or upon the first signs of possible or actual exposure to infection or disease (e.g., due to exposure to or infection with neisseria).
Screening method
In one example, a method of assessing the effectiveness of fHbp in a subject in a vaccine composition comprises: (a) immunizing a host animal (e.g., a non-human mammalian host animal, e.g., a rodent, e.g., a mouse) with a composition comprising a fHbp of the invention; and (b) detecting the level of bactericidal antibodies in the host. The subject methods can further include assessing the susceptibility of the host animal to a bacterial pathogen, and administering to the host animal a vaccine comprising the subject fHbp.
In another example, the method can include preparing and identifying antibodies elicited by fHbp in the subject. The method comprises isolating antibodies having binding affinity for fHbp from a host animal, contacting a bacterial cell with the isolated antibodies; and assessing binding of the antibody to the bacterial cell. Additional steps may include assessing competitive binding of antibodies to fHbp to human factor H; when the antibody is administered to an animal with a bacterial pathogen, bactericidal activity against the bacterial pathogen is assessed. In certain embodiments, the antibody is in a population of antibodies, and the method can further comprise: isolating one or more antibodies that bind to the bacterial cells from the population of antibodies. A distinctive aspect is that the isolated antibody is bactericidal against bacterial cells, e.g., by complement-mediated bactericidal activity and/or phagocytic activity, which can reduce the viability of the bacteria in human blood.
Particularly preferred bacterial pathogens are any or all variant groups of the various capsular groups of Neisseria meningitidis, such as Neisseria meningitidis serogroup B, Neisseria meningitidis serogroup C, Neisseria meningitidis serogroup X, Neisseria meningitidis serogroup Y, Neisseria meningitidis serogroup W-135, and the like.
Evaluation pairFHBPMethod of answering
The invention provides methods of determining the likelihood that an fHbp will elicit a bactericidal response in an individual; and methods of evaluating fHbp variants suitable for addition to an immunogenic composition.
Determining the likelihood that fHbp will elicit a bactericidal response
The present invention provides a method of determining the likelihood that a fHbp (e.g., a fHbp present in a neisseria vaccine) will elicit a bactericidal response in an individual against at least one neisseria meningitidis strain. The methods generally include determining the ability of an antibody, which is present in serum obtained from an individual immunized with fHbp, to inhibit binding of fH to fHbp. The antibody inhibits binding of fH to fHbp at a level at least about 10% greater, at least about 25% greater, at least about 50% greater, at least about 75% greater, at least about 2-fold greater, at least about 10-fold greater, at least about 50-fold greater, at least about 100-fold greater, or more than 100-fold greater than the level of inhibition of binding of fH to fHbp by a control antibody that inhibits binding of fH to fHbp but does not produce a bactericidal response, indicating that fHbp is likely to elicit a bactericidal response against at least one neisseria meningitidis strain. In certain embodiments, the fHbp is a non-naturally occurring fHbp that has a lower affinity for human factor H (fH) than fHbp ID1, as described above.
The extent to which the fHbp variant elicits antibody inhibition of binding of fH to fHbp can be determined using the assays described herein, or any other known assay. For example, the fH and/or the fHbp can comprise a detectable label, and inhibition of binding of the fH/fHbp can be determined by detecting the amount of the labeled component present in the fH/fHbp complex, and/or by detecting the amount of label present in free fH and/or free fHbp (e.g., fH or fHbp that is not in the fH/fHbp complex).
In one example, a detection method to assess binding of fH to fHbp includes using fHbp immobilized on a support (e.g., fHbp immobilized in a well of an elisa plate). A mixture containing a fixed concentration of human fH and a dilution of the antibody to be tested (e.g., antisera, e.g., from a human or non-human test animal (e.g., a mouse) that received a challenge dose of the antibody of the immunogenic composition) is added to the wells and incubated for a time sufficient for the antibody to bind. After washing the wells, bound fH is detected using a specific anti-fH antiserum (e.g., goat or donkey) containing a labeled component, or a second labeled antibody (e.g., rabbit anti-goat or anti-donkey antiserum). Percent inhibition of binding to fH can be calculated by the amount of bound fH without the addition of human or mouse antibody.
In another variation of such assays, binding of fH to live bacteria in the presence or absence of the antisera to be tested is assessed using flow cytometry. Bacterial cells are incubated with fixed concentrations of fH (e.g., detectably labeled fH) and different dilutions of test serum containing antibodies. The bacteria are washed and bound fH is detected (e.g., as described above).
Thus, instead of directly assessing the bactericidal activity of antisera from an individual immunized with fHbp for their ability to inhibit fH/fHbp binding, the antisera can be assessed. The methods of the invention for determining the likelihood that an fHbp will elicit a bactericidal response in an individual can provide information to a clinician or other medical personnel to determine whether a particular immunogenic composition has elicited an effective bactericidal response in an individual.
Immunized individuals were tested by ELISA and had similar serum IgG anti-fHbp antibody titers. If antisera provide better inhibition of fH binding overall, it is indicated that a more effective, higher quality anti-fHbp antibody response is generated and will provide better protection. Thus, for example, when comparing the anti-neisserial antibody responses produced by two individuals (by comparing anti-fHbp antibodies, i.e., inhibition of a 1:10,000 serum dilution compared to other individuals at a 1:3000 dilution), an individual with higher inhibitory activity will have a higher quality of anti-fHbp antibodies, which will provide better protection. Thus, fH inhibition assays can be used as an alternative to complement-mediated bactericidal titer assays, which typically take more time and are more difficult to detect than fH inhibition.
Evaluating fHbp variants
The present invention provides methods for assessing or predicting the likelihood that a fHbp variant will be effective in eliciting a bactericidal antibody response in an individual. The methods generally include evaluating the ability of an antibody specific for a fHbp variant to inhibit binding of fH to fHbp. The inhibition of binding of fH to fHbp by antibodies elicited by immunization with a fHbp variant is positively correlated with the bactericidal activity of the antibodies elicited by the fHbp variant. Antibodies raised against fHbp variants may be considered suitable vaccine candidates for eliciting protection against one or more neisserial strains if they inhibit binding of fH to fHbp at higher serum dilutions.
For example, the invention provides a method of determining the likelihood that a non-naturally occurring fHbp that has a lower affinity for human fH than fHbp ID1 will elicit bactericidal antibodies in an individual against at least one neisseria meningitidis strain. The methods generally include determining the ability of an antibody elicited by a non-naturally occurring fHbp to inhibit binding of fH to the fHbp in a non-human subject animal. The level of inhibition of binding of fH to fHbp by the non-naturally occurring fHbp by the antibody is at least about 10% greater, at least about 25% greater, at least about 50% greater, at least about 75% greater, at least about 2-fold greater, at least about 10-fold greater, at least about 50-fold greater, at least about 100-fold greater, or greater than 100-fold greater than the level of inhibition of binding of fH to fHbp by the antibody elicited by fHbp ID1 in the non-human subject animal, indicating that the non-naturally occurring fHbp is likely to elicit a bactericidal response against at least one neisseria meningitidis strain.
Suitable non-human test animals include, for example, mice, rats, rabbits, and the like. The extent to which the fHbp variant elicits antibody inhibition of binding of fH to fHbp can be determined using the assays described herein, or any other known assay. The bactericidal activity of the antibody can be readily determined using the assay described herein, or any other known assay.
For a given non-naturally occurring fHbp having a lower affinity for human fH than fHbp ID1, a method of determining its likelihood of eliciting bactericidal antibodies in an individual against at least one neisseria meningitidis strain may be used to identify suitable immunogens (and/or eliminate unsuitable immunogens), for example during vaccine development.
Examples
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited in this application are incorporated by reference in their entirety for all purposes.
- 上一篇：一种医用注射器针头装配设备
- 下一篇：一种分泌型格氏环肽及其制备方法