NZ747917B2 - Neisseria meningitidis compositions and methods thereof - Google Patents

Neisseria meningitidis compositions and methods thereof

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Publication number
NZ747917B2
NZ747917B2 NZ747917A NZ74791713A NZ747917B2 NZ 747917 B2 NZ747917 B2 NZ 747917B2 NZ 747917 A NZ747917 A NZ 747917A NZ 74791713 A NZ74791713 A NZ 74791713A NZ 747917 B2 NZ747917 B2 NZ 747917B2
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NZ
New Zealand
Prior art keywords
seq
polypeptide
lipidated
amino acid
variant
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Application number
NZ747917A
Other versions
NZ747917A (en
Inventor
Annaliesa Sybil Anderson
Susan Kay Hoiseth
Kathrin Ute Jansen
Justin Keith Moran
Mark E Ruppen
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Pfizer Inc
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Application filed by Pfizer Inc filed Critical Pfizer Inc
Publication of NZ747917A publication Critical patent/NZ747917A/en
Publication of NZ747917B2 publication Critical patent/NZ747917B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6018—Lipids, e.g. in lipopeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/095—Neisseria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/22—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Neisseriaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1217—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Neisseriaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C12N15/1031—Mutagenizing nucleic acids mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/36—Neisseria

Abstract

Disclosed is an isolated ORF2086 polypeptide consisting of the amino acid sequence SEQ ID NO: 66. Also disclosed is the use of an immunogenic composition comprising an isolated non-lipidated, non-pyruvylated ORF2086 polypeptide having the amino acid sequence consisting of SEQ ID NO: 66 in the manufacture of a medicament for eliciting a bactericidal antibody against Neisseria meningitidis serogroup C in a mammal. ufacture of a medicament for eliciting a bactericidal antibody against Neisseria meningitidis serogroup C in a mammal.

Description

NEISSERIA ITIDIS COMPOSITIONS AND S THEREOF This application is a divisional application out of New Zealand patent application 731330, itself a divisional application out of New Zealand patent application 718108, itself a onal application out of New d patent application 628449, all dated 6 March 2013, each of which is incorporated herein by reference.
FIELD OF THE INVENTION The present ion relates to Neisseria meningitidis compositions and methods ng thereto.
BACKGROUND OF THE INVENTION Neisseria meningitids is a Gram-negative encapsulated bacterium that can cause sepsis, itis and death. N. meningitidis can be classified into about 13 serogroups ding serogroups A, B, C, E29, H, I, K, L, W-135, X , Y and Z) based on chemically and antigenically distinctive polysaccharide capsules. Five of the serogroups (A, B, C, Y, and W135) are responsible for the majority of disease. ococcal meningitis is a devastating disease that can kill children and young adults within hours despite the availability of otics. There is a need for improved immunogenic compositions against meningococcal serogroups A, B, C, Y, and W135 and/or X.
It is an object of the present invention to go some way to meeting this need, and/or to at least provide the public with a useful choice.
In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.
SUMMARY OF THE INVENTION In a first aspect, the present invention provides an isolated polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 66.
In a second aspect, the present ion provides an immunogenic composition sing the polypeptide of the first aspect.
In a third aspect, the present invention provides an ed nucleic acid encoding an isolated polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 66.
In a fourth aspect, the present invention provides a method of inducing an immune response against Neisseria meningitidis in a mammal excluding a human being comprising administering to the mammal an effective amount of an immunogenic composition comprising an isolated polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 66.
In a fifth aspect, the present invention provides a method of eliciting a bactericidal antibody against Neisseria meningitidis in a mammal ing a human being comprising administering to the mammal an effective amount of an immunogenic composition comprising an isolated polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 66.
In a sixth aspect, the present ion provides an immunogenic composition comprising an isolated pidated, non- pyruvylated ORF2086 polypeptide from Neisseria meningitidis oup B, and at least one conjugate selected from: a) a conjugate of a capsular saccharide of Neisseria meningitidis serogroup A, b) a conjugate of a capsular saccharide of ria meningitidis serogroup C, c) a conjugate of a capsular saccharide of Neisseria itidis serogroup W135; and d) a ate of a capsular saccharide of Neisseria meningitidis serogroup Y, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO: 66.
In a seventh aspect, the t invention provides use of an immunogenic composition according to the second aspect or sixth aspect in the manufacture of a medicament for inducing an immune response against Neisseria meningitidis in a mammal.
In an eighth aspect, the present invention provides use of an immunogenic composition according to the second aspect or sixth aspect in the manufacture of a medicament for eliciting a icidal dy against Neisseria itidis in a mammal.
Described herein are Neisseria meningitidis compositions and methods thereof.
Described herein is an isolated polypeptide including an amino acid sequence that is at least 95% identical to SEQ ID NO: 71, wherein the first twenty amino acid residues of the sequence does not contain a cysteine.
In one embodiment, the ed polypeptide includes the amino acid sequence at positions 1-184 of SEQ ID NO: 71.
In one ment, the isolated polypeptide includes the amino acid ce at positions 158-185 of SEQ ID NO: 71. In another embodiment, the isolated polypeptide includes the amino acid sequence at positions 159-186 of SEQ ID NO: 71.
In one embodiment, the isolated polypeptide includes at least 6 contiguous amino acids from the amino acid sequence at positions 185-254 of SEQ ID NO: 71.
In one embodiment, the isolated polypeptide is non-pyruvylated.
In one embodiment, the isolated polypeptide is non-lipidated.
In one embodiment, the isolated polypeptide is immunogenic.
In one embodiment, the isolated polypeptide includes the amino acid sequence consisting of the sequence set forth in SEQ ID NO: 71.
Described herein is an isolated polypeptide including an amino acid sequence that is at least 95% identical to SEQ ID NO: 76, n the first twenty amino acid residues of the sequence does not contain a cysteine.
In one embodiment, the isolated polypeptide includes the amino acid sequence SEQ ID NO: 76.
In one embodiment, the isolated ptide includes the amino acid sequence SEQ ID NO: 76, wherein the cysteine at position 1 is deleted. In another embodiment, the isolated polypeptide includes the amino acid sequence SEQ ID NO: 76, n the cysteine at position 1 is substituted with an amino acid that is not a Cys residue. In one ment, the isolated polypeptide includes the amino acid sequence SEQ ID NO: In one embodiment, the isolated polypeptide is non-pyruvylated. In one embodiment, the isolated polypeptide is non-lipidated. In one embodiment, the isolated polypeptide is immunogenic.
Described herein is an immunogenic composition including the polypeptide as in any of the embodiments aforementioned. Described herein is an immunogenic composition including the polypeptide as in any of the embodiments described herein.
Described herein is an isolated nucleic acid ce encoding an isolated polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 71.
In one embodiment, the isolated nucleic acid sequence includes SEQ ID NO: 72.
Described herein is an genic composition including an isolated nonlipidated , non-pyruvylated ORF2086 polypeptide from Neisseria meningitidis serogroup B, and at least one conjugate selected from: a) a conjugate of a capsular saccharide of Neisseria meningitidis serogroup A; b) a ate of a capsular saccharide of ria meningitidis serogroup C; c) a conjugate of a capsular saccharide of Neisseria meningitidis serogroup W135; and d) a conjugate of a capsular saccharide of Neisseria meningitidis serogroup Y.
In one ment, the immunogenic composition includes at least two ates selected from: a) a conjugate of a capsular ride of Neisseria meningitidis serogroup A; b) a conjugate of a capsular saccharide of Neisseria meningitidis serogroup C; c) a conjugate of a capsular saccharide of Neisseria meningitidis serogroup W135; and d) a conjugate of a capsular ride of Neisseria meningitidis oup Y.
In one embodiment, the immunogenic composition es at least three conjugates selected from: a) a conjugate of a capsular ride of Neisseria itidis serogroup A; b) a conjugate of a capsular saccharide of Neisseria meningitidis serogroup C; c) a conjugate of a capsular saccharide of Neisseria itidis serogroup W135; and d) a conjugate of a capsular saccharide of Neisseria itidis serogroup Y.
In one embodiment, the immunogenic composition includes a conjugate of a capsular saccharide of Neisseria meningitidis serogroup A; a conjugate of a capsular ride of Neisseria meningitidis serogroup C; a conjugate of a capsular saccharide of Neisseria meningitidis serogroup W135; and a ate of a capsular saccharide of Neisseria meningitidis serogroup Y.
In one embodiment, the polypeptide is a subfamily A polypeptide.
In one ment, the polypeptide is a subfamily B polypeptide.
In one embodiment, the polypeptide is a non-pyruvylated non-lipidated A05.
In one ment, the polypeptide is a non-pyruvylated non-lipidated A12.
In one embodiment, the polypeptide is a non-pyruvylated non-lipidated A22.
In one embodiment, the polypeptide is a non-pyruvylated non-lipidated B01.
In one embodiment, the ptide is a non-pyruvylated non-lipidated B09.
In one ment, the polypeptide is a non-pyruvylated non-lipidated B44.
In one embodiment, the polypeptide is a non-pyruvylated non-lipidated B22.
In one embodiment, the polypeptide is a ruvylated non-lipidated B24.
In one embodiment, the polypeptide is a ruvylated non-lipidated A62.
In one embodiment, the polypeptide includes the amino acid sequence selected from the group ting of SEQ ID NO: 44, SEQ ID NO: 49, SEQ ID NO: 55, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 71, and SEQ ID NO: 75. In one embodiment, the polypeptide includes the amino acid sequence SEQ ID NO: 77.
Described herein is a method of inducing an immune se against Neisseria meningitidis in a mammal. The method includes administering to the mammal an effective amount of an immunogenic composition including an ed non-lipidated, non-pyruvylated ORF2086 polypeptide from Neisseria meningitidis serogroup B, and at least one conjugate selected from: a) a conjugate of a capsular saccharide of Neisseria meningitidis serogroup A; b) a conjugate of a capsular saccharide of Neisseria itidis serogroup C; c) a conjugate of a capsular saccharide of Neisseria meningitidis serogroup W135; and d) a conjugate of a capsular saccharide of ria meningitidis serogroup Y.
Described herein is a method of eliciting a bactericidal antibody against Neisseria meningitidis serogroup C in a mammal. The method in cludes administering to the mammal an ive amount of an immunogenic composition including an isolated nonlipidated , non-pyruvylated ORF2086 polypeptide from Neisseria meningitidis serogroup In one ment, the polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 71 or the amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, wherein the cysteine at position 1 is d. In r embodiment, the polypeptide includes the amino acid sequence set forth in SEQ ID NO: 76. In yet another embodiment, the cysteine at position 1 of the ptide is deleted. In a further embodiment, the polypeptide includes the amino acid sequence set forth in SEQ ID NO: 77.
In one embodiment, the immunogenic composition further includes at least one conjugate selected from: a) a conjugate of a capsular saccharide of Neisseria meningitidis serogroup A; b) a conjugate of a ar saccharide of Neisseria itidis serogroup C; c) a conjugate of a capsular saccharide of Neisseria meningitidis serogroup W135; and d) a conjugate of a capsular saccharide of Neisseria meningitidis serogroup Y.
Described herein is a method of ing a bactericidal antibody against Neisseria meningitidis serogroup Y in a mammal. The method includes stering to the mammal an effective amount of an immunogenic composition including an an isolated non-lipidated, non-pyruvylated 6 polypeptide from Neisseria meningitidis serogroup B.
In one embodiment, the polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 71 or the amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, wherein the cysteine at position 1 is deleted. In another embodiment, the polypeptide includes the amino acid sequence set forth in SEQ ID NO: 76. In yet another embodiment, the cysteine at position 1 of the polypeptide is deleted. In a further ment, the polypeptide includes the amino acid sequence set forth in SEQ ID NO: 77.
In one ment, the immunogenic composition further es at least one conjugate selected from: a) a conjugate of a capsular saccharide of Neisseria meningitidis serogroup A; b) a conjugate of a capsular saccharide of Neisseria meningitidis serogroup C; c) a conjugate of a capsular saccharide of Neisseria meningitidis oup W135; and d) a conjugate of a capsular saccharide of Neisseria meningitidis serogroup Y.
Described herein is a method of eliciting a bactericidal antibody against Neisseria meningitidis in a mammal, ing administering to the mammal an effective amount of an immunogenic composition including an isolated non-lipidated, non-pyruvylated 6 polypeptide from Neisseria meningitidis serogroup B, and at least one conjugate selected from: a) a conjugate of a capsular saccharide of Neisseria meningitidis serogroup A; b) a conjugate of a capsular saccharide of Neisseria meningitidis oup C; c) a conjugate of a capsular saccharide of Neisseria meningitidis serogroup W135; and d) a conjugate of a capsular saccharide of Neisseria itidis serogroup Y.
The term “comprising” as used in this specification and claims means “consisting at least in part of”. When interpreting statements in this specification and claims which e the term “comprising”, other features besides the features prefaced by this term in each statement can also be present. Related terms such as “comprise” and “comprised” are to be interpreted in similar manner.
In the description in this specification reference may be made to subject matter which is not within the scope of the claims of the current ation. That subject matter should be y identifiable by a person skilled in the art and may assist in putting into practice the invention as defined in the claims of this ation.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1: P2086 Variant Nucleic Acid Sequences.
Figure 2: P2086 Variant Amino Acid Sequences. The r stalk in the N-terminal tail of each variant is underlined.
Figure 3: Structure of the ORF2086 n Figure 4: Removal of N-terminal Cys Results in Loss of sion in E. coli.
Figure 5: Effect of Gly/Ser Stalk Length on Non-lipidated ORF2086 Variant Expression.
The sequence ated with the protein variant labeled B01 is set forth in SEQ ID NO: . The sequence associated with the protein variant labeled B44 is set forth in SEQ ID NO: 36. The sequence associated with the n variant labeled A05 is set forth in SEQ ID NO: 37. The sequence associated with the protein variant labeled A22 is set forth in SEQ ID NO: 38. The sequence associated with the protein variant labeled B22 is set forth in SEQ ID NO: 39. The sequence associated with the protein variant labeled A19 is set forth in SEQ ID NO: 40.
Figure 6: High Levels of Non-lipidated B09 Expression Despite A Short r Stalk.
The left two lanes demonstrated expression of the N-terminal Cys-deleted B09 variant before and after induction. The third and fourth lanes demonstrate expression of the N-terminal Cys positive B09 variant before and after induction. The right most lane is a molecular weight standard. The amino acid sequence shown under the image is set forth in SEQ ID NO: 41. The nucleotide sequence representative of the N-terminal Cysdeleted A22 variant, referred to as “A22_001” in the figure, is set forth in SEQ ID NO: 42, which is shown under SEQ ID NO: 41 in the figure. The nucleotide sequence entative of the N-terminal Cys-deleted B22 variant, referred to as “B22_001” in the figure, is set forth in SEQ ID NO: 52. The nucleotide sequence representative of the N-terminal Cys-deleted B09 variant, referred to as “B09_004” in the figure, is set forth in SEQ ID NO: 53.
Figure 7: Codon zation Increases Expression of Non-lipidated B22 and A22 Variants. The left panel demonstrates expression of the N-terminal leted B22 variant before (lanes 1 and 3) and after (lanes 2 and 4) IPTG induction. The right panel demonstrates expression of the N-terminal Cys-deleted A22 variant before (lane 7) and after (lane 8) IPTG ion. Lanes 5 and 6 are molecular weight standards.
Figure 8: P2086 t Nucleic and Amino Acid Sequences Figure 9A-9B: Sequence alignment of selected wild-type subfamily A and B fHBP variants discussed in es 15-19. Note that the N nus of A62 is 100% identical to B09 and its C-terminus is 100% identical to A22. The sequences shown are A05 (SEQ ID NO: 13); A12 (SEQ ID NO: 14); A22 (SEQ ID NO: 15); A62 (SEQ ID NO: 70); B09 (SEQ ID NO: 18); B24 (SEQ ID NO: 20); and Consensus (SEQ ID NO: 78).
SEQUENCE IDENTIFIERS SEQ ID NO: 1 sets forth a DNA sequence for the N. meningitidis, serogroup B, 2086 variant A04 gene, which includes a codon encoding an N-terminal Cys.
SEQ ID NO: 2 sets forth a DNA sequence for the N. meningitidis, serogroup B, 2086 variant A05 gene, which includes a codon encoding an N-terminal Cys.
SEQ ID NO: 3 sets forth a DNA sequence for the N. itidis, serogroup B, 2086 variant A12 gene, which includes a codon encoding an N-terminal Cys.
SEQ ID NO: 4 sets forth a DNA sequence for the N. meningitidis, serogroup B, 2086 variant A12-2 gene, which includes a codon encoding an inal Cys.
SEQ ID NO: 5 sets forth a DNA sequence for the N. meningitidis, serogroup B, 2086 variant A22 gene, which includes a codon encoding an N-terminal Cys.
SEQ ID NO: 6 sets forth a DNA sequence for the N. meningitidis, serogroup B, 2086 variant B02 gene, which includes a codon encoding an N-terminal Cys.
SEQ ID NO: 7 sets forth a DNA sequence for the N. meningitidis, serogroup B, 2086 variant B03 gene, which es a codon encoding an N-terminal Cys.
SEQ ID NO: 8 sets forth a DNA sequence for the N. meningitidis, serogroup B, 2086 variant B09 gene, which includes a codon encoding an N-terminal Cys.
SEQ ID NO: 9 sets forth a DNA sequence for the N. meningitidis, serogroup B, 2086 variant B22 gene, which includes a codon encoding an N-terminal Cys.
SEQ ID NO: 10 sets forth a DNA sequence for the N. itidis, oup B, 2086 variant B24 gene, which es a codon encoding an N-terminal Cys.
SEQ ID NO: 11 sets forth a DNA sequence for the N. meningitidis, serogroup B, 2086 variant B44 gene, which includes a codon ng an N-terminal Cys.
SEQ ID NO: 12 sets forth the amino acid sequence for the N. meningitidis, serogroup B, 2086 variant A04, which es an N-terminal Cys at amino acid position 1.
SEQ ID NO: 13 sets forth the amino acid sequence for the N. meningitidis, serogroup B, 2086 variant A05, which includes an N-terminal Cys at amino acid position 1.
SEQ ID NO: 14 sets forth the amino acid sequence for the N. meningitidis, serogroup B, 2086 variant A12, which includes an N-terminal Cys at amino acid position 1.
SEQ ID NO: 15 sets forth the amino acid sequence for the N. meningitidis, serogroup B, 2086 variant A22, which includes an N-terminal Cys at amino acid position 1.
SEQ ID NO: 16 sets forth the amino acid sequence for the N. meningitidis, serogroup B, 2086 t B02, which includes an N-terminal Cys at amino acid position 1.
SEQ ID NO: 17 sets forth the amino acid sequence for the N. meningitidis, serogroup B, 2086 variant B03, which includes an N-terminal Cys at amino acid position 1.
SEQ ID NO: 18 sets forth the amino acid sequence for the N. meningitidis, serogroup B, 2086 variant B09, which includes an N-terminal Cys at amino acid position 1.
SEQ ID NO: 19 sets forth the amino acid sequence for the N. itidis, serogroup B, 2086 variant B22, which includes an N-terminal Cys at amino acid position 1.
SEQ ID NO: 20 sets forth the amino acid sequence for the N. meningitidis, serogroup B, 2086 variant B24, which includes an N-terminal Cys at amino acid position 1.
SEQ ID NO: 21 sets forth the amino acid sequence for the N. itidis, serogroup B, 2086 variant B44, which includes an N-terminal Cys at amino acid position 1.
SEQ ID NO: 22 sets forth a DNA sequence for a forward primer, shown in Example 2.
SEQ ID NO: 23 sets forth a DNA sequence for a e primer, shown in Example 2.
SEQ ID NO: 24 sets forth a DNA sequence for a forward primer, shown in e 2, Table 1.
SEQ ID NO: 25 sets forth a DNA sequence for a e primer, shown in Example 2, Table 1.
SEQ ID NO: 26 sets forth a DNA sequence for a forward primer, shown in Example 2, Table 1.
SEQ ID NO: 27 sets forth a DNA sequence for a reverse primer, shown in Example 2, Table 1.
SEQ ID NO: 28 sets forth a DNA sequence for a r stalk, shown in Example 4.
SEQ ID NO: 29 sets forth the amino acid sequence for a Gly/Ser stalk, shown in Example 4, which is encoded by, for example SEQ ID NO: 28.
SEQ ID NO: 30 sets forth a DNA sequence for a Gly/Ser stalk, shown in e 4.
SEQ ID NO: 31 sets forth the amino acid sequence a Gly/Ser stalk, shown in Example 4, which is encoded by, for e SEQ ID NO: 30.
SEQ ID NO: 32 sets forth a DNA sequence for a Gly/Ser stalk, shown in Example 4.
SEQ ID NO: 33 sets forth the amino acid sequence for a Gly/Ser stalk, which is encoded by, for example, SEQ ID NO: 32 and SEQ ID NO: 34.
SEQ ID NO: 34 sets forth a DNA ce for a Gly/Ser stalk, shown in Example 4.
SEQ ID NO: 35 sets forth the amino acid sequence for the N-terminus of N. meningitidis, serogroup B, 2086 variant B01, shown in Figure 5.
SEQ ID NO: 36 sets forth the amino acid sequence for the N-terminus of N. meningitidis, serogroup B, 2086 variant B44, shown in Figure 5.
SEQ ID NO: 37 sets forth the amino acid sequence for the N-terminus of N. meningitidis, serogroup B, 2086 variant A05, shown in Figure 5.
SEQ ID NO: 38 sets forth the amino acid sequence for the N-terminus of N. meningitidis, serogroup B, 2086 variant A22, shown in Figure 5.
SEQ ID NO: 39 sets forth the amino acid sequence for the N-terminus of N. meningitidis, serogroup B, 2086 variant B22, shown in Figure 5.
SEQ ID NO: 40 sets forth the amino acid sequence for the N-terminus of N. itidis, serogroup B, 2086 variant A19, shown in Figure 5.
SEQ ID NO: 41 sets forth the amino acid sequence for the N-terminus of a N. meningitidis, serogroup B, 2086 variant, shown in Figure 6.
SEQ ID NO: 42 sets forth a DNA sequence for the N-terminus of N. meningitidis, serogroup B, 2086 variant A22, shown in Figure 6.
SEQ ID NO: 43 sets forth a codon-optimized DNA sequence for the N. meningitidis, serogroup B, 2086 variant B44 gene, wherein the codon encoding an N-terminal cysteine is deleted, as compared to SEQ ID NO: 11. Plasmid pDK087 includes SEQ ID NO: 43.
SEQ ID NO: 44 sets forth the amino acid sequence for a non-lipidated N. meningitidis, serogroup B, 2086 variant B44. SEQ ID NO: 44 is cal to SEQ ID NO: 21 wherein the N-terminal cysteine at position 1 of SEQ ID NO: 21 is deleted. SEQ ID 44 is encoded by, for e, SEQ ID NO: 43.
SEQ ID NO: 45 sets forth a codon-optimized DNA sequence for the N. meningitidis, oup B, 2086 variant B09 gene, wherein the codon encoding an N-terminal cysteine is deleted, and wherein the sequence includes codons encoding an onal Gly/Ser region, as compared to SEQ ID NO: 8. Plasmid pEB063 includes SEQ ID NO: SEQ ID NO: 46 sets forth a codon-optimized DNA sequence for the N. itidis, serogroup B, 2086 variant B09 gene, wherein the codon encoding an N-terminal cysteine is deleted, as compared to SEQ ID NO: 8. Plasmid pEB064 includes SEQ ID NO: 46.
SEQ ID NO: 47 sets forth a codon-optimized DNA sequence for the N. meningitidis, serogroup B, 2086 variant B09 gene, wherein the codon encoding an N-terminal cysteine is deleted, as ed to SEQ ID NO: 8. Plasmid pEB 065 includes SEQ ID NO: 47.
SEQ ID NO: 48 sets forth a DNA sequence for the N. meningitidis, serogroup B, 2086 variant B09 gene, wherein the codon encoding an N-terminal cysteine is deleted, as compared to SEQ ID NO: 8. d pLA134 includes SEQ ID NO: 48.
SEQ ID NO: 49 sets forth the amino acid sequence for a non-lipidated N. meningitidis, serogroup B, 2086 variant B09. SEQ ID NO: 49 is identical to SEQ ID NO: 18 n the N-terminal cysteine at position 1 of SEQ ID NO: 18 is deleted. SEQ ID 49 is encoded by, for example, a DNA sequence selected from the group consisting of SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48.
SEQ ID NO: 50 sets forth the amino acid sequence for the N. meningitidis, serogroup B, 2086 variant B09, n the codon encoding an N-terminal cysteine is d and wherein the sequence includes codons encoding an additional r region, as compared to SEQ ID NO: 18. SEQ ID NO: 50 is encoded by, for example, SEQ ID NO: SEQ ID NO: 51 sets forth a DNA sequence for the N. meningitidis, serogroup B, 2086 t B44 gene, wherein the codon encoding an N-terminal cysteine is deleted, as compared to SEQ ID NO: 11. d pLN056 includes SEQ ID NO: 51.
SEQ ID NO: 52 sets forth a DNA ce for the N-terminus of N. meningitidis, serogroup B, 2086 variant B22, shown in Figure 6.
SEQ ID NO: 53 sets forth a DNA sequence for the N-terminus of N. meningitidis, serogroup B, 2086 variant B09, shown in Figure 6.
SEQ ID NO: 54 sets forth a DNA sequence for a N. meningitidis, serogroup B, 2086 variant A05 gene, wherein the codon encoding an N-terminal cysteine is deleted, as compared to SEQ ID NO: 2.
SEQ ID NO: 55 sets forth the amino acid sequence for a non-lipidated N. meningitidis, serogroup B, 2086 variant A05. SEQ ID NO: 55 is identical to SEQ ID NO: 13 wherein the N-terminal cysteine at position 1 of SEQ ID NO: 13 is deleted. SEQ ID NO: 55 is encoded by, for example, SEQ ID NO: 54.
SEQ ID NO: 56 sets forth the amino acid sequence of a serine-glycine repeat sequence, shown in Example 7.
SEQ ID NO: 57 sets forth the amino acid sequence for a non-lipidated N. meningitidis, serogroup B, 2086 variant B01. SEQ ID NO: 57 is identical to SEQ ID NO: 58 wherein the N-terminal ne at position 1 of SEQ ID NO: 58 is d.
SEQ ID NO: 58 sets forth the amino acid sequence for the N. meningitidis, oup B, 2086 variant B01, which includes an N-terminal Cys at amino acid position 1.
SEQ ID NO: 59 sets forth the amino acid sequence for the N. meningitidis, serogroup B, 2086 variant B15, which includes an N-terminal Cys at amino acid position 1.
SEQ ID NO: 60 sets forth the amino acid sequence for the N. meningitidis, serogroup B, 2086 variant B16, which es an N-terminal Cys at amino acid position 1.
SEQ ID NO: 61 sets forth a DNA sequence for the N. meningitidis, serogroup B, 2086 variant B22, in which the codon for the N-terminal Cys at amino acid position 1 of SEQ ID NO: 19 is replaced with a codon for a e.
SEQ ID NO: 62 sets forth the amino acid sequence for the N. meningitidis, serogroup B, 2086 t B22, in which the N-terminal Cys at amino acid position 1 of SEQ ID NO: 19 is replaced with a Glycine.
SEQ ID NO: 63 sets forth a DNA sequence for the N. meningitidis, serogroup B, 2086 variant A22, in which the codon for the N-terminal Cys at amino acid position 1 of SEQ ID NO: 15 is replaced with a codon for a Glycine.
SEQ ID NO: 64 sets forth the amino acid sequence for the N. meningitidis, serogroup B, 2086 variant A22, in which the N-terminal Cys at amino acid position 1 of SEQ ID NO: 15 is replaced with a Glycine.
SEQ ID NO: 65 sets forth a codon-optimized DNA sequence (pEB042) encoding a nonlipidated , non-pyruvylated A05 polypeptide.
SEQ ID NO: 66 sets forth the amino acid ce for a non-lipidated N. meningitidis, serogroup B, 2086 variant A12. SEQ ID NO: 66 is identical to SEQ ID NO: 14 wherein the N-terminal cysteine at position 1 of SEQ ID NO: 14 is deleted. SEQ ID NO: 66 is d by, for example, SEQ ID NO: 67.
SEQ ID NO: 67 sets forth a codon-optimized DNA sequence for a non-lipidated, non- pyruvylated A12 polypeptide.
SEQ ID NO: 68 sets forth the amino acid sequence for a non-lipidated N. meningitidis, serogroup B, 2086 variant A22. SEQ ID NO: 68 is identical to SEQ ID NO: 15 wherein the N-terminal cysteine at position 1 of SEQ ID NO: 15 is deleted. SEQ ID NO: 68 is encoded by, for example, SEQ ID NO: 69.
SEQ ID NO: 69 sets forth a codon-optimized DNA ce for a non-lipidated, nonpyruvylated A22 polypeptide.
SEQ ID NO: 70 sets forth the amino acid sequence for the N. meningitidis serogroup B, 2086 variant A62, which includes an N-terminal Cys at amino acid position 1.
SEQ ID NO: 71 sets forth the amino acid sequence for a non-lipidated N. meningitidis, serogroup B, 2086 variant A62. SEQ ID NO: 71 is identical to SEQ ID NO: 70 wherein the inal cysteine at position 1 of SEQ ID NO: 70 is deleted.
SEQ ID NO: 72 sets forth a optimized DNA sequence for SEQ ID NO: 71.
SEQ ID NO: 73 sets forth a optimized DNA ce (pDK086) for a N. meningitidis, serogroup B, 2086 variant A05 gene, wherein the codon encoding an N- terminal cysteine is deleted, as compared to SEQ ID NO: 2.
SEQ ID NO: 74 sets forth the amino acid sequence for the N. meningitidis, serogroup B, 2086 variant A29, which includes an N-terminal Cys at amino acid position 1.
SEQ ID NO: 75 sets forth the amino acid sequence for a non-lipidated N. meningitidis, serogroup B, 2086 variant B22. SEQ ID NO: 75 is identical to SEQ ID NO: 19 wherein the N-terminal cysteine at on 1 of SEQ ID NO: 19 is deleted.
SEQ ID NO: 76 sets forth the amino acid sequence for a N. meningitidis, oup B, 2086 variant A05.
SEQ ID NO: 77 sets forth the amino acid sequence for a non-lipidated N. meningitidis, serogroup B, 2086 variant A05. SEQ ID NO: 77 is cal to SEQ ID NO: 19 wherein the N-terminal cysteine at position 1 of SEQ ID NO: 76 is not present.
SEQ ID NO: 78 sets forth the amino acid sequence for a consensus sequence shown in -9B.
SEQ ID NO: 79 is identical to SEQ ID NO: 78 except that the Cys at position 1 of SEQ ID NO: 78 is not t.
SEQ ID NO: 80 sets forth the amino acid sequence for the N. meningitidis, serogroup B, 2086 variant B24. SEQ ID NO: 80 is identical to SEQ ID NO: 20 wherein the N-terminal cysteine at position 1 of SEQ ID NO: 20 is deleted.
SEQ ID NO: 81 sets forth the amino acid sequence for the N. itidis, serogroup B, 2086 variant B24. SEQ ID NO: 81 is identical to SEQ ID NO: 20 wherein the residues at positions 1-3 of SEQ ID NO: 20 are deleted.
DETAILED DESCRIPTION OF THE INVENTION Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable s and materials are described below. The als, methods and examples are illustrative only, and are not intended to be limiting. All publications, patents and other documents mentioned herein are incorporated by nce in their entirety.
Definitions The term "antigen" lly refers to a biological molecule, usually a protein, peptide, polysaccharide, lipid or conjugate which contains at least one epitope to which a cognate antibody can selectively bind; or in some instances to an immunogenic substance that can stimulate the tion of antibodies or T-cell responses, or both, in an , including compositions that are injected or absorbed into an animal. The immune response may be generated to the whole le, or to one or more various portions of the molecule (e.g., an epitope or hapten). The term may be used to refer to an individual molecule or to a homogeneous or heterogeneous population of antigenic molecules. An antigen is recognized by antibodies, T-cell receptors or other elements of specific humoral and/or cellular immunity. The term "antigen" includes all related antigenic epitopes. Epitopes of a given antigen can be identified using any number of epitope mapping techniques, well known in the art. See, e.g., Epitope Mapping ols in s in Molecular Biology, Vol. 66 (Glenn E. , Ed., 1996) Humana Press, Totowa, N. J. For example, linear epitopes may be determined by e.g., rently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports. Such techniques are known in the art and described in, e.g., U.S. Pat. No. 871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81:3998-4002; Geysen et al. (1986) Molec. Immunol. 23:709-715, all incorporated herein by reference in their entireties. Similarly, conformational epitopes may be fied by determining spatial conformation of amino acids such as by, e.g., x-ray crystallography and nsional nuclear magnetic resonance. See, e.g., Epitope Mapping ols, supra. Furt hermore, for purposes of the present invention, an "antigen" may also be used to refer to a protein that includes modifications, such as deletions, additions and substitutions (generally conservative in nature, but they may be non-conservative), to the native sequence, so long as the protein maintains the ability to elicit an immunological se. These modifications may be deliberate, as through irected mutagenesis, or through particular synthetic procedures, or through a genetic engineering ch, or may be accidental, such as through mutations of hosts, which produce the antigens. Furthermore, the antigen can be derived, obtained, or isolated from a microbe, e.g. a ium, or can be a whole organism. Similarly, an oligonucleotide or polynucleotide, which expresses an n, such as in nucleic acid immunization applications, is also included in the definition.
Synthetic antigens are also included, for example, itopes, flanking epitopes, and other recombinant or synthetically derived antigens (Bergmann et al. (1993) Eur. J.
Immunol. 23:2777 2781; Bergmann et al. (1996) J. Immunol. 157:3242 3249; Suhrbier, A. (1997) l. and Cell Biol. 75:402 408; Gardner et al. (1998) 12th World AIDS Conference, Geneva, rland, Jun. 28 - Jul. 3, 1998).
The term "conservative" amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility hydrophobicity, hilicity, and/or the amphipathic nature of the residues involved. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, , proline, tryptophan, and methionine; polar/neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include ne, lysine, and ine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. In some embodiments, the conservative amino acid changes alter the primary sequence of the ORF2086 polypeptides, but do not alter the function of the molecule. When generating these mutants, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a polypeptide is generally tood in the art (Kyte & tle, 1982, J. Mol. Biol., 157(1):105-32). It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still result in a polypeptide with similar biological activity. Each amino acid has been assigned a athic index on the basis of its hydrophobicity and charge characteristics. Those indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine ; cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); ine ; aspartate (-3.5); asparagine (-3.5); lysine ; and arginine (-4.5).
It is believed that the relative hydropathic ter of the amino acid residue determines the secondary and tertiary structure of the resultant polypeptide, which in turn defines the interaction of the polypeptide with other molecules, such as enzymes, ates, receptors, antibodies, antigens, and the like. It is known in the art that an amino acid can be substituted by another amino acid having a similar hydropathic index and still obtain a functionally lent polypeptide. In such changes, the substitution of amino acids whose hydropathic indices are within +/-2 is preferred, those within +/-1 are particularly preferred, and those within +/-0.5 are even more particularly preferred.
Conservative amino acids tutions or insertions can also be made on the basis of hydrophilicity. As bed in U.S. Pat. No. 4,554,101, which is hereby incorporated by reference the greatest local average hydrophilicity of a polypeptide, as governed by the hydrophilicity of its adjacent amino acids, ates with its immunogenicity and antigenicity, i.e., with a biological property of the polypeptide. U.S.
Pat. No. 4,554,101reciates that the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine ; aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); proline (-0.5±1); threonine (-0.4); alanine (-0.5); ine (-0.5); cysteine (-1.0); nine (-1.3); valine ; leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); phan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent polypeptide. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred; those within ±1 are particularly red; and those within ±0.5 are even more particularly preferred.
Exemplary substitutions which take various of the foregoing teristics into consideration are well known to those of skill in the art and include, without limitation: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
The term "effective immunogenic amount" as used herein refers to an amount of a polypeptide or composition comprising a polypeptide which is effective in eliciting an immune response in a vertebrate host. For example, an effective immunogenic amount of a rLP2086 protein of this invention is an amount that is effective in eliciting an immune response in a vertebrate host. The particular "effective immunogenic dosage or amount" will depend upon the age, weight and medical condition of the host, as well as on the method of administration. Suitable doses are readily determined by persons skilled in the art.
The term "Gly/Ser stalk" as used herein refers to the series of Gly and Ser residues immediately downstream of the N-terminal Cys residue of a protein encoded by ORF2086. There can be n 5 and 12 Gly and Ser residues in the Gly/Ser stalk. Accordingly, the r stalk consists of amino acids 2 to between 7 and 13 of the protein encoded by ORF2086. Preferably, the Gly/Ser stalk consists of amino acids 2 and up to between 7 and 13 of the protein encoded by ORF2086. The r stalks of the P2086 variants of the present invention are represented by the underlined sequences in Figure 2 (SEQ ID NO: 12-21). As shown herein, the length of the r stalk can affect the stability or expression level of a non-lipidated P2086 variant. In an exemplary embodiment, effects from affecting the length of the Gly/Ser stalk are compared to those from the corresponding wild-type variant.
The term "immunogenic" refers to the ability of an antigen or a vaccine to elicit an immune response, either humoral or cell-mediated, or both.
An "immunogenic amount", or an "immunologically ive amount" or "dose", each of which is used interchangeably herein, generally refers to the amount of antigen or immunogenic composition ient to elicit an immune response, either a cellular (T cell) or humoral (B cell or antibody) response, or both, as measured by standard assays known to one skilled in the art.
The term "immunogenic composition" s to any pharmaceutical composition containing an antigen, e.g. a microorganism, or a component thereof, which composition can be used to elicit an immune response in a subject. The genic compositions of the present invention can be used to treat a human susceptible to N. meningidis ion, by means of administering the genic compositions via a systemic transdermal or l route. These administrations can include injection via the intramuscular (i.m.), intraperitoneal (i.p.), intradermal (i.d.) or subcutaneous routes; application by a patch or other transdermal delivery device; or via mucosal administration to the oral/alimentary, respiratory or genitourinary tracts. In one embodiment, the immunogenic composition may be used in the manufacture of a vaccine or in the elicitation of a polyclonal or onal antibodies that could be used to passively protect or treat a subject.
Optimal amounts of components for a particular immunogenic composition can be ascertained by standard studies involving observation of appropriate immune responses in subjects. Following an initial vaccination, subjects can receive one or l booster immunizations adequately spaced.
The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring or from it's host sm if it is a recombinant , or taken from one environment to a ent environment). For example, an "isolated" protein or peptide is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is d, or ntially free of chemical precursors or other chemicals when chemically synthesized, or otherwise present in a mixture as part of a chemical reaction. In the present invention, the proteins may be isolated from the bacterial cell or from cellular debris, so that they are provided in a form useful in the manufacture of an immunogenic composition. The term "isolated" or "isolating" may include purifying, or purification, including for example, the methods of purification of the proteins, as described herein. The language "substantially free of cellular material" includes preparations of a polypeptide or protein in which the polypeptide or n is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, a n or peptide that is substantially free of cellular material includes preparations of the capsule polysaccharide, protein or peptide having less than about 30%, 20%, 10%, 5%, 2.5%, or 1%, (by dry ) of contaminating n or polysaccharide or other cellular al. When the polypeptide/protein is recombinantly ed, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation. When polypeptide or n is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein or polysaccharide. Accordingly, such preparations of the ptide or n have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than polypeptide/protein or polysaccharide nt of interest.
The term minal tail" as used herein refers to the N-terminal portion of a protein encoded by ORF2086, which attaches the protein to the cell membrane. An N-terminal tail is shown at the bottom of the side view structure in Figure 3. An N-terminal tail typically ses the N-terminal 16 amino acids of the n encoded by ORF2086. In some embodiments, the N-terminal tail is amino acids 1-16 of any one of SEQ ID NOs: 12-21.The term "ORF2086" as used herein refers to Open Reading Frame 2086 from a ria species ia. Neisseria ORF2086, the proteins encoded therefrom, fragments of those proteins, and immunogenic compositions comprising those proteins are known in the art and are bed, e.g., in WO2003/063766, and in U.S. Patent Application Publication Nos. US 57413 and US 20090202593, each of which is hereby incorporated by reference in its entirety.
The term “P2086” generally refers to the protein d by ORF2086. The “P” before “2086” is an abbreviation for “protein.” The P2086 proteins described herein may be lipidated or non-lipidated. “LP2086” and “P2086” typically refer to lipidated and non-lipidated forms of a 2086 protein, respectively. The P2086 n described herein may be recombinant. “rLP2086” and “rP2086” typically refer to lipidated and non-lipidated forms of a recombinant 2086 protein, respectively. “2086” is also known as factor H-binding protein (fHBP) due to its ability to bind to factor H.
The term "pharmaceutically acceptable diluent, excipient, and/or carrier" as used herein is ed to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with administration to humans or other vertebrate hosts. Typically, a pharmaceutically acceptable diluent, excipient, and/or carrier is a diluent, excipient, and/or carrier approved by a regulatory agency of a Federal, a state government, or other regulatory agency, or listed in the U.S. Pharmacopeia or other generally ized pharmacopeia for use in animals, including humans as well as non-human mammals. The term diluent, excipient, and/or "carrier" refers to a t, adjuvant, excipient, or vehicle with which the ceutical composition is administered. Such pharmaceutical diluent, excipient, and/or carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic . Water, saline solutions and aqueous dextrose and glycerol solutions can be employed as liquid diluents, excipients, and/or carriers, particularly for injectable solutions. le pharmaceutical diluents and/or excipients include , glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
The composition, if desired, can also contain minor s of wetting, bulking, fying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, ned release formulations and the like.
Examples of suitable pharmaceutical diluent, excipient, and/or carriers are described in gton's Pharmaceutical Sciences" by E. W. Martin. The formulation should suit the mode of administration. The appropriate diluent, excipient, and/or carrier will be evident to those skilled in the art and will depend in large part upon the route of administration.
A "protective" immune response refers to the ability of an immunogenic composition to elicit an immune response, either l or cell mediated, which serves to protect the subject from an ion. The protection provided need not be absolute, i.e., the infection need not be totally prevented or eradicated, if there is a statistically significant improvement compared with a control population of subjects, e.g. infected s not administered the vaccine or immunogenic ition. Protection may be limited to mitigating the severity or rapidity of onset of symptoms of the infection. In general, a "protective immune response" would include the induction of an increase in antibody levels specific for a particular antigen in at least 50% of subjects, ing some level of measurable functional antibody ses to each n. In particular situations, a "protective immune response" could include the induction of a two fold increase in antibody levels or a four fold se in antibody levels specific for a particular antigen in at least 50% of subjects, including some level of measurable functional antibody responses to each antigen. In certain embodiments, opsonising dies correlate with a protective immune response. Thus, protective immune response may be assayed by measuring the percent decrease in the bacterial count in a serum bactericidal activity (SBA) assay or an opsonophagocytosis assay, for instance those described below. Such assays are also known in the art. For meningococcal vaccines, for example, the SBA assay is an established surrogate for protection. In some embodiments, there is a decrease in bacterial count of at least 10%, 25%, 50%, 65%, 75%, 80%, 85%, 90%, 95% or more, as ed to the bacterial count in the absence of the immunogenic composition.
The terms "protein", eptide" and "peptide" refer to a polymer of amino acid residues and are not limited to a minimum length of the product. Thus, peptides, oligopeptides, dimers, multimers, and the like, are included within the definition. Both ength proteins and fragments thereof are encompassed by the definition. The terms also e modifications, such as deletions, additions and substitutions (generally conservative in nature, but which may be non-conservative), to a native sequence, preferably such that the protein maintains the ability to elicit an immunological response within an animal to which the protein is administered. Also included are post-expression modifications, e.g. glycosylation, acetylation, lipidation, phosphorylation and the like.
Active variants and nts of the sed polynucleotides and polypeptides are also described herein. "Variants" refer to substantially similar sequences. As used , a "variant polypeptide" refers to a polypeptide derived from the native protein by a modification of one or more amino acids at the N-terminal and/or C-terminal end of the native protein. The modification may include deletion (so-called truncation) of one or more amino acids at the N-terminal and/or C-terminal end of the native protein; deletion and/or addition of one or more amino acids at one or more internal sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Variant ptides continue to possess the desired biological activity of the native polypeptide, that is, they are immunogenic. A variant of an polypeptide or polynucleotide ce disclosed herein (i.e. SEQ ID NOS: 1-25 or 39) will typically have at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more ce identity with the nce sequence.
The term ent" refers to a portion of an amino acid or nucleotide sequence comprising a specified number of contiguous amino acid or nucleotide residues. In particular embodiments, a fragment of a polypeptide disclosed herein may retain the biological activity of the full-length polypeptide and hence be immunogenic. Fragments of a polynucleotide may encode protein fragments that retain the biological ty of the protein and hence be immunogenic. Alternatively, fragments of a polynucleotide that are useful as PCR primers generally do not retain biological ty. Thus, fragments of a nucleotide sequence disclosed herein may range from at least about 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175, 200, 225, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 contiguous nucleotides or up to the full-length polynucleotide. Fragments of a polypeptide sequence disclosed herein may comprise at least 10, 15, 20, 25, 30, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 400, 425, 450, 475, or 500 contiguous amino acids, or up to the total number of amino acids present in the fulllength polypeptide.
The term "recombinant" as used herein refers to any n, polypeptide, or cell expressing a gene of interest that is produced by genetic ering methods. The term "recombinant" as used with respect to a protein or polypeptide, means a polypeptide produced by sion of a recombinant polynucleotide. The proteins described hereinmay be isolated from a natural source or produced by genetic engineering methods. "Recombinant," as used herein, further bes a nucleic acid molecule, which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide with which it is associated in nature. The term "recombinant" as used with respect to a host cell means a host cell which includes a recombinant polynucleotide.
The term ct" refers to a mammal, bird, fish, reptile, or any other animal.
The term "subject" also includes humans. The term "subject" also includes household pets. Non-limiting examples of old pets include: dogs, cats, pigs, rabbits, rats, mice, s, rs, guinea pigs, s, birds, snakes, lizards, fish, turtles, and frogs. The term "subject" also includes ock animals. Non-limiting examples of livestock animals include: alpaca, bison, camel, cattle, deer, pigs, horses, llamas, mules, donkeys, sheep, goats, rabbits, reindeer, yak, chickens, geese, and turkeys.
The term “mammals” as used herein refers to any mammal, such as, for example, humans, mice, rabbits, non-human primates. In a preferred embodiment, the mammal is a human.
The terms "vaccine" or "vaccine composition", which are used hangeably, refer to pharmaceutical compositions comprising at least one immunogenic composition that induces an immune response in a subject.
General Description Described herein are previously unidentified ulties expressing non-lipidated P2086 variants and provides methods for overcoming these difficulties and novel compositions therefrom. While plasmid constructs encoding non-lipidated P2086 variants provided strong expression of the non-lipidated variants, these variants were pyruvylated on the N-terminal Cys. Pyruvylation prevents or reduces the likelihood of cturing consistency or uniformity of the polypeptides. The inventors further found that deletion of the N-terminal Cys from the non-lipidated P2086 variant sequences avoided pyruvylation of non-lipidated P2086 variants. Attempts to overcome the lation by deletion of the codon for the N-terminal Cys either abrogated expression or resulted in the expression of insoluble variants. Alternatively, removal of the N-terminal Cys from the non-lipidated P2086 variants decreased sion in some ts. Surprisingly, however, the inventors discovered that at least nonpyruvylated non-lipidated A05, A12, A22, A62, B01, B09, B22, and B44 variants can be sed despite deletion of the N-terminal Cys residue. Generally, these polypeptides could be expressed without additional modifications other than the Cys deletion, as ed to the corresponding wild-type non-lipidated sequence. See, for example, Examples 2 and 4. rmore, the inventors discovered that the non-pyruvylated non-lipidated variants were singly immunogenic and they unexpectedly elicited bactericidal dies.
Accordingly, described herein are two methods for overcoming or reducing the likelihood of these difficulties in expressing pidated variants. r, additional methods are contemplated herein. The first method was to vary the length of the Gly/Ser stalk in the N-terminal tail, immediately downstream of the N-terminal Cys. The second method was codon optimization within the N-terminal tail. However, optimization of additional codons is plated herein. These methods provide enhanced expression of soluble non-lipidated P2086 variants. For example, in one embodiment, enhanced expression of e non-lipidated P2086 variants is compared to expression of the corresponding wild-type non-lipidated variants.
Isolated ptides The inventors surprisingly discovered isolated non-pyruvylated, non-lipidated ORF2086 polypeptides. The inventors further discovered that the polypeptides are unexpectedly immunogenic and are capable of eliciting a bactericidal immune response.
As used herein, the term yruvylated” refers to a polypeptide having no pyruvate content. Non-lipidated ORF2086 polypeptides having a pyruvate content typically exhibited a mass shift of +70, as compared to the corresponding ype polypeptide. In one embodiment, the inventive polypeptide does not exhibit a mass shift of +70 as compared to the corresponding wild-type non-lipidated polypeptide when measured by mass spectrometry. See, for example, Example 10.
In another embodiment, the isolated non-pyruvylated, non-lipidated ORF2086 polypeptide includes a on of an N-terminal cysteine residue compared to the corresponding wild-type non-lipidated ORF2086 polypeptide. The term “N-terminal cysteine” refers to a cysteine (Cys) at the N-terminal or N-terminal tail of a polypeptide.
More specifically, the “N-terminal cysteine” as used herein refers to the N-terminal cysteine at which LP2086 lipoproteins are lipidated with a tripalmitoyl lipid tail, as is known in the art. For example, when referring to any one of SEQ ID NOs: 12-21 as a reference sequence, the N-terminal cysteine is located at position 1. As another example, when referring to SEQ ID NO: 70 as a reference sequence, the N-terminal cysteine is d at position 1.
The term “wild-type non-lipidated 6 polypeptide” or type nonlipidated 2086 polypeptide” or “wild-type non-lipidated polypeptide” as used herein refers to an ORF2086 polypeptide having an amino acid ce that is identical to the amino acid sequence of the corresponding mature lipidated ORF2086 ptide found in nature. The only difference n the non-lipidated and lipidated molecules is that the wild-type non-lipidated ORF2086 polypeptide is not lipidated with a tripalmitoyl lipid tail at the inal cysteine.
As is known in the art, the pidated 2086 form is produced by a protein lacking the original leader sequence or by a leader sequence which is replaced with a portion of ce that does not specify a site for fatty acid acylation in a host cell.
See, for example, WO2003/063766, which is incorporated herein by reference in its entirety.
Examples of a non-lipidated ORF2086 e not only a wild-type non-lipidated ORF2086 polypeptide just described but also polypeptides having an amino acid sequence according to any one of SEQ ID NOs: 12-21 wherein the N-terminal Cys is deleted and ptides having an amino acid sequence according to any one of SEQ ID NOs: 12-21 wherein the N-terminal Cys is substituted with an amino acid that is not a Cys residue. Another example of a non-lipidated ORF2086 polypeptide includes a polypeptide having an amino acid sequence according to SEQ ID NO: 70 wherein the N-terminal Cys is deleted and a polypeptide having an amino acid sequence according to SEQ ID NO: 70 wherein the N-terminal Cys is substituted with an amino acid that is not a Cys residue. Further examples of a non-lipidated ORF2086 polypeptide include amino acid sequences selected from SEQ ID NO: 44 (B44), SEQ ID NO: 49 (B09), SEQ ID NO: 55 (A05), SEQ ID NO: 57 (B01), SEQ ID NO: 58 (B01), SEQ ID NO: 62 (B22), SEQ ID NO: 64 (A22), and SEQ ID NO: 75 (B22). Yet r es of a nonlipidated 6 ptide include amino acid ces selected from SEQ ID NO: 66 (A12), SEQ ID NO: 68 (A22), and SEQ ID NO: 71 (A62). More examples include SEQ ID NO: 80 (B24) and SEQ ID NO: 81 (B24). Additional examples of a idated ORF2086 polypeptide include the amino acid sequences set forth in SEQ ID NO: 76 and SEQ ID NO: 77. In one embodiment, the non-lipidated polypeptide includes the amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence encoding the corresponding non-lipidated polypeptide. For example, in an exemplary embodiment, the non-lipidated A62 polypeptide includes the amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 71.
Examples of a wild-type non-lipidated ORF2086 polypeptide include ptides having an amino acid sequence ing to any one of SEQ ID NOs: 12-21, shown in Figure 2, SEQ ID NO: 58, SEQ ID NO: 59 , and SEQ ID NO: 60. Another example of a wild-type non-lipidated ORF2086 polypeptide includes a polypeptide having the amino acid sequence according to SEQ ID NO: 70. These ary wild-type non-lipidated ORF2086 polypeptides include an N-terminal Cys.
As used herein, for example, a “non-lipidated” B44 polypeptide includes a polypeptide having the amino acid sequence selected from SEQ ID NO: 21, SEQ ID NO: 21 wherein the N-terminal Cys at position 1 is deleted, and SEQ ID NO: 44. A “wild-type non-lipidated” B44 polypeptide includes a polypeptide having the amino acid sequence SEQ ID NO: 21. A yruvylated non-lipidated” B44 ptide includes a polypeptide having the amino acid sequence selected from SEQ ID NO: 21 wherein the N-terminal Cys at position 1 is deleted, and SEQ ID NO: 44.
As another example, as used herein, a “non-lipidated” B09 polypeptide es a polypeptide having the amino acid ce selected from SEQ ID NO: 18, SEQ ID NO: 18 wherein the N-terminal Cys at position 1 is deleted, SEQ ID NO: 49, and SEQ ID NO: 50. A “wild-type non-lipidated” B09 polypeptide includes a polypeptide having the amino acid sequence SEQ ID NO: 18. A “non-pyruvylated non-lipidated” B09 includes a polypeptide having the amino acid sequence selected from SEQ ID NO: 18 wherein the N-terminal Cys at position 1 is deleted, SEQ ID NO: 49, and SEQ ID NO: As yet a further e, as used , a “non-lipidated” A05 polypeptide includes a polypeptide having the amino acid sequence selected from SEQ ID NO: 13, SEQ ID NO: 13 n the N-terminal Cys at position 1 is deleted, and SEQ ID NO: 55. Another example of a ipidated” A05 polypeptide includes a polypeptide having the amino acid sequence selected from SEQ ID NO: 13 wherein the N-terminal Cys at position 1 is substituted with an amino acid that is not a Cys residue. An additional example of a “non-lipidated” A05 polypeptide includes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 76. Yet another example of a “non-lipidated” A05 ptide includes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 77. A “wild-type pidated” A05 includes a polypeptide having the amino acid sequence SEQ ID NO: 13. A yruvylated non-lipidated” A05 includes a polypeptide having the amino acid sequence selected from SEQ ID NO: 13 wherein the N-terminal Cys at position 1 is deleted and SEQ ID NO: 55. Further examples of a “non-pyruvylated non-lipidated” A05 includes a polypeptide having the amino acid sequence selected from SEQ ID NO: 13 wherein the N-terminal Cys at position 1 is substituted with an amino acid that is not a Cys residue; SEQ ID NO: 76 wherein the Cys at on 1 is deleted; SEQ ID NO: 76 wherein the Cys at position 1 is substituted with an amino acid that is not a Cys residue; and SEQ ID NO: 77.
As used herein, a “non-lipidated” A62 polypeptide includes a polypeptide having the amino acid ce selected from SEQ ID NO: 70, SEQ ID NO: 70 wherein the N- terminal Cys at position 1 is deleted, and SEQ ID NO: 71. r example of a nonlipidated A62 polypeptide includes a polypeptide having SEQ ID NO: 70 wherein the N- terminal Cys at position 1 is substituted with an amino acid that is not a Cys residue. A “wild-type non-lipidated” A62 polypeptide includes a polypeptide having the amino acid sequence SEQ ID NO: 70. A “non-pyruvylated non-lipidated” A62 includes a ptide having the amino acid sequence selected from SEQ ID NO: 70 wherein the N-terminal Cys at position 1 is deleted, and SEQ ID NO: 71. Another example of a nonpyruvylated non-lipidated A62 polypeptide includes a polypeptide having SEQ ID NO: 70 wherein the N-terminal Cys at position 1 is substituted with an amino acid that is not a Cys residue. Preferably, a “non-pyruvylated pidated” A62 includes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 71.
As used herein, a “non-lipidated” A12 ptide es a polypeptide having the amino acid ce selected from SEQ ID NO: 14, SEQ ID NO: 14 wherein the N- terminal Cys at on 1 is deleted, and SEQ ID NO: 66. A “wild-type non-lipidated” A12 polypeptide includes a polypeptide having the amino acid sequence SEQ ID NO: 14. A “non-pyruvylated non-lipidated” A12 includes a polypeptide having the amino acid sequence selected from SEQ ID NO: 14 wherein the N-terminal Cys at position 1 is d, and SEQ ID NO: 66.
As used herein, a “non-lipidated” A22 polypeptide es a polypeptide having the amino acid sequence selected from SEQ ID NO: 15, SEQ ID NO: 15 wherein the N- terminal Cys at on 1 is deleted, SEQ ID NO: 64, and SEQ ID NO: 68. A “wild-type non-lipidated” A22 polypeptide includes a polypeptide having the amino acid sequence SEQ ID NO: 15. A “non-pyruvylated non-lipidated” A22 includes a polypeptide having the amino acid sequence selected from SEQ ID NO: 15 wherein the N-terminal Cys at position 1 is deleted, SEQ ID NO: 64, and SEQ ID NO: 68. Preferably, a “nonpyruvylated non-lipidated” A22 includes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 68.
The term “deletion” of the N-terminal Cys as used herein includes a mutation that deletes the N-terminal Cys, as ed to a wild-type non-lipidated ptide sequence. For example, a “deletion” of the N-terminal Cys refers to a removal of the amino acid Cys from a reference sequence, e.g., from the corresponding ype sequence, thereby resulting in a decrease of an amino acid residue as compared to the nce sequence. Unless otherwise described, the terms “N-terminal Cys,” “N- terminal Cys at position 1,” “Cys at position 1” are interchangeable.
In r embodiment, the N-terminal Cys is substituted with an amino acid that is not a Cys residue. For example, in an exemplary embodiment, the N-terminal Cys at position 1 of SEQ ID NOs: 12-21 includes a C→G substitution at position 1. See, for example, SEQ ID NO: 62 as compared to SEQ ID NO: 19 (B22 wild-type), and SEQ ID NO: 64 as compared to SEQ ID NO: 15 (A22 wild-type). Exemplary amino acids to replace the N-terminal Cys include any non-Cys amino acid, preferably a polar uncharged amino acid such as, for example, glycine. In a preferred embodiment, the substitution is made with a non-conservative e to Cys.
The inventors surprisingly discovered that expressing non-lipidated ORF2086 polypeptides having a deletion of an N-terminal Cys residue resulted in no detectable pyruvylation when measured by mass spectrometry, as compared to the ponding wild-type pidated ORF2086 polypeptide. Examples of non-pyruvylated non- ted ORF2086 polypeptides include those having an amino acid sequence selected from the group consisting of SEQ ID NO:12 (A04), SEQ ID NO:13 (A05), SEQ ID NO:14 (A12), SEQ ID NO:15 (A22), SEQ ID NO:16 (B02)¸ SEQ ID NO:17 (B03), SEQ ID NO:18 (B09), SEQ ID NO:19 (B22), SEQ ID NO: 20 (B24), SEQ ID NO: 21 (B44), and SEQ ID NO: 70 (A62), wherein the ne at position 1 is deleted. Another example of a non-pyruvylated non-lipidated ORF2086 polypeptide includes a polypeptide having the amino acid sequence SEQ ID NO: 58 (B01), wherein the cysteine at position 1 is deleted. onal examples of isolated non-pyruvylated, non-lipidated ORF2086 polypeptides include ptides having an amino acid sequence selected from the group consisting of SEQ ID NO: 44 , SEQ ID NO: 49, SEQ ID NO: 50 , SEQ ID NO: 55, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 71, and SEQ ID NO: 75. A further e of a non-pyruvylated non-lipidated 6 polypeptide includes a polypeptide having the amino acid sequence SEQ ID NO: 57 (B01). r example of an isolated non-pyruvylated non-lipidated ORF2086 ptide includes a polypeptide having SEQ ID NO: 77 (A05); a polypeptide having SEQ ID NO: 76 (A05) wherein the Cys at position 1 is deleted; and a polypeptide having SEQ ID NO: 76 (A05) wherein the Cys at position 1 is substituted with an amino acid that is not a Cys residue.
Further examples of non-pyruvylated non-lipidated ORF2086 polypeptides include those having an amino acid sequence selected from the group consisting of SEQ ID NO:12 (A04), SEQ ID NO:13 (A05), SEQ ID NO:14 (A12), SEQ ID NO:15 (A22), SEQ ID NO: 58 (B01), SEQ ID NO:16 (B02)¸ SEQ ID NO:17 (B03), SEQ ID NO:18 (B09), SEQ ID NO:19 (B22), SEQ ID NO: 20 (B24), SEQ ID NO: 21 (B44), and SEQ ID NO: 70 (A62) wherein the cysteine at position 1 is tuted with an amino acid that is not a Cys e. Preferably, the non-pyruvylated non-lipidated 2086 polypeptide includes at least about 250, 255, or 260 consecutive amino acids, and at most about 270, 269, 268, 267, 266, 265, 264, 263, 260, 259, 258, 257, 256, or 255 consecutive amino acids. Any minimum value may be combined with any maximum value to define a range. More preferably, the polypeptide has at least 254 or 262 consecutive amino acids. In some embodiments, the polypeptide has at most 262 consecutive amino acids. In other embodiments, the polypeptide has at most 254 consecutive amino acids. In one embodiment, the non-pyruvylated non-lipidated polypeptide includes the amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% cal to a ce encoding the corresponding non-pyruvylated non-lipidated polypeptide. For example, in an exemplary embodiment, the non-pyruvylated pidated A62 polypeptide includes the amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 71.
In one embodiment, the isolated non-pyruvylated, non-lipidated ORF2086 polypeptide is encoded by a tide ce that is operatively linked to an expression system, wherein the expression system is capable of being expressed in a bacterial cell. In an exemplary embodiment, the nucleotide sequence is linked to a regulatory sequence that controls expression of the nucleotide sequence.
Suitable expression systems, tory sequences, and bacterial cells are known in the art. For example, any plasmid expression vector, e.g., PET™ (Novogen, Madison Wis.) or PMAL™ (New England Biolabs, Beverly, Mass.) can be used as long as the polypeptide is able to be expressed in a bacterial cell. Preferably, the PET™ vector is used for cloning and expression of recombinant proteins in E. coli. In the PET™ system, the cloned gene may be expressed under the control of a phage T7 promotor. Exemplary bacterial cells include Pseudomonas scens, and preferably, E. coli.
Described herein is a non-pyruvylated non-lipidated ORF2086 polypeptide obtainable by the process. The polypeptide is preferably ed. Also described herein are compositions that include a non-pyruvylated non-lipidated ORF2086 ptide able by a process. The ition is preferably an genic composition. The process includes expressing a nucleotide sequence encoding a polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16¸ SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 58, and SEQ ID NO: 70, n the cysteine at position 1 is deleted. In another embodiment, the process includes expressing a tide sequence encoding a polypeptide having the amino acid sequence SEQ ID NO: 76, wherein the cysteine at position 1 is deleted. In a further embodiment, the process includes expressing a nucleotide sequence encoding a polypeptide having the amino acid sequence ed from the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16¸ SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO: , SEQ ID NO: 21, SEQ ID NO: 58, and SEQ ID NO: 70, wherein the cysteine at position 1 is substituted with an amino acid that is not a Cys residue. The nucleotide sequence is operatively linked to an expression system that is capable of being expressed in a bacterial cell.
In one embodiment, the process includes sing a nucleotide sequence encoding a polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO: 44, SEQ ID NO: 49 , SEQ ID NO: 50, SEQ ID NO: 55, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 71, SEQ ID NO: 57, and SEQ ID NO: 75. In another embodiment, the process includes expressing a nucleotide sequence encoding a polypeptide having the amino acid sequence SEQ ID NO: 77. In another embodiment, the nucleotide sequence is selected from the group consisting of SEQ ID NO: 43, SEQ ID NO: 51, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 45, SEQ ID NO: 54, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, and SEQ ID NO: 72. Preferably the bacterial cell is E. coli.
B09, B44, A05: Described herein is a composition that includes a first ed polypeptide, which includes the amino acid sequence set forth in SEQ ID NO: 49 (B09), and a second isolated polypeptide, which includes the amino acid sequence set forth in SEQ ID NO: 44 (B44). In a preferred embodiment, the polypeptides are immunogenic.
In r preferred ment, the composition further includes an ORF2086 subfamily A polypeptide from serogroup B N. meningitidis. Preferably, the ORF2086 subfamily A polypeptide is a non-pyruvylated non-lipidated ORF2086 subfamily A polypeptide. In an exemplary embodiment, the 6 subfamily A polypeptide is A05, examples of which include, for example, SEQ ID NO: 13, wherein the N-terminal cysteine at on 1 is deleted, and SEQ ID NO: 55. In another exemplary embodiment, the composition includes a non-pyruvylated non-lipidated A05 polypeptide having the amino acid sequence SEQ ID NO: 76 wherein the Cys at position 1 is deleted; SEQ ID NO: 76 n the Cys at on 1 is substituted with an amino acid that is not a Cys residue; and SEQ ID NO: 77.
Polypeptide domains Described herein is a method for producing an isolated polypeptide. The method includes expressing in a bacterial cell a polypeptide, which es a sequence having greater than 90% identity to SEQ ID NO:21, said sequence includes at least one domain selected from the group consisting of amino acids 13-18 of SEQ ID NO: 21, amino acids 21-34 of SEQ ID NO: 21, and amino acids 70-80 of SEQ ID NO: 21, or a ation thereof, wherein the polypeptide lacks an N-terminal cysteine. The method further es purifying the polypeptide. The polypeptide produced therein includes a non-pyruvylated non-lipidated ORF2086 ptide. Preferably, the polypeptide is immunogenic. In a preferred embodiment, the bacterial cell is E. coli.
Examples of polypeptides that include at least one domain selected from the group consisting of amino acids 13-18 of SEQ ID NO: 21, amino acids 21-34 of SEQ ID NO: 21, and amino acids 70-80 of SEQ ID NO: 21, or a combination thereof, include SEQ ID NO: 12 (A04), SEQ ID NO: 13 (A05), SEQ ID NO: 14 (A12), SEQ ID NO: 15 (A22), SEQ ID NO: 16 (B02), SEQ ID NO: 17 (B03), SEQ ID NO: 18 (B09), SEQ ID NO: 19 (B22), SEQ ID NO: 20 (B24), and SEQ ID NO: 21 (B44). Preferably the ne at position 1 of these polypeptides is deleted. In another embodiment, the cysteine at position 1 is substituted with an amino acid that is not a Cys residue. Further exemplary polypeptides include SEQ ID NO: 44, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 55, SEQ ID NO: 62, and SEQ ID NO: 64. Another exemplary polypeptide includes SEQ ID NO: 70 and SEQ ID NO: 71. A further exemplary polypeptide includes SEQ ID NO: 76.
Yet another exemplary polypeptide includes SEQ ID NO: 77. Additional examples include SEQ ID NO: 80 (B24) and SEQ ID NO: 81 (B24).
In one exemplary embodiment, the isolated polypeptide sequence further includes at least one domain selected from the group consisting of amino acids 96-116 of SEQ ID NO: 21, amino acids 158-170 of SEQ ID NO: 21, amino acids 172-185 of SEQ ID NO: 21, amino acids 187-199 of SEQ ID NO: 21, amino acids 213-224 of SEQ ID NO: 21, amino acids 226-237 of SEQ ID NO: 21, amino acids 239-248 of SEQ ID NO: 21, or a combination f. Examples of polypeptides that include at least one domain selected from the group consisting of amino acids 13-18 of SEQ ID NO: 21, amino acids 21-34 of SEQ ID NO: 21, and amino acids 70-80 of SEQ ID NO: 21, or a combination thereof, and further including at least one domain selected from the group consisting of amino acids 96-116 of SEQ ID NO: 21, amino acids 0 of SEQ ID NO: 21, amino acids 172-185 of SEQ ID NO: 21, amino acids 187-199 of SEQ ID NO: 21, amino acids 213-224 of SEQ ID NO: 21, amino acids 226-237 of SEQ ID NO: 21, amino acids 239-248 of SEQ ID NO: 21, or a combination thereof, include SEQ ID NO: 16 (B02), SEQ ID NO: 17 (B03), SEQ ID NO: 18 (B09), SEQ ID NO: 19 (B22), SEQ ID NO: 20 (B24), and SEQ ID NO: 21 (B44). Preferably the cysteine at position 1 of these polypeptides is deleted. Further exemplary polypeptides include a polypeptide having the amino acid sequence selected from SEQ ID NO: 44, SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 55, and SEQ ID NO: 62.
Described herein is an isolated polypeptide produced by a process described herein. In one embodiment, the isolated polypeptide is a non-pyruvylated non-lipidated ptide. Also bed is an immunogenic composition produced by a s described .
Nucleotide sequences encoding the polypeptides B09: Described herein is an isolated polypeptide that es the amino acid sequence set forth in SEQ ID NO: 18 wherein the N-terminal Cys at position 1 is d or SEQ ID NO: 49. Exemplary tide sequences that encode SEQ ID NO: 49 include sequences selected from SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48.
Preferably, the nucleotide sequence is SEQ ID NO: 46. Described herein is an isolated nucleotide sequence that includes SEQ ID NO: 46. Described herein is an isolated nucleotide sequence that es SEQ ID NO: 47. Described herein is an isolated nucleotide sequence that includes SEQ ID NO: 48.
Described herein is a plasmid including a nucleotide sequence selected from SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, and SEQ ID NO: 45, wherein the d is capable of being expressed in a bacterial cell. Suitable expression s, regulatory sequences, and bacterial cells are known in the art, as described above.
Preferably, the bacterial cell is E. coli.
Described herein is an isolated polypeptide that includes the amino acid sequence set forth in SEQ ID NO: 50. In an exemplary embodiment, SEQ ID NO: 50 is encoded by SEQ ID NO: 45.
B44: Described herein is an isolated polypeptide that includes the amino acid sequence set forth in SEQ ID NO: 21 n the N-terminal Cys is deleted or SEQ ID NO: 44. Exemplary nucleotide sequences that encode SEQ ID NO: 44 include sequences selected from SEQ ID NO: 43 and SEQ ID NO: 51. Preferably, the nucleotide sequence is SEQ ID NO: 43. Also described herein is an isolated nucleotide ce that includes SEQ ID NO: 43.
A05: Described herein is an isolated polypeptide that includes the amino acid sequence set forth in SEQ ID NO: 13 (A05) wherein the N-terminal Cys at position 1 is deleted or SEQ ID NO: 55. Exemplary nucleotide sequences that encode SEQ ID NO: 55 include sequences selected from SEQ ID NO: 54, SEQ ID NO: 65, and SEQ ID NO: 73. Preferably, the nucleotide sequence is SEQ ID NO: 65. Described herein is an isolated nucleotide sequence that includes SEQ ID NO: 54. Described herein is an isolated nucleotide sequence that includes SEQ ID NO: 65. Also described herein is an isolated tide sequence that includes SEQ ID NO: 73.
A12: Described herein is an isolated polypeptide that includes the amino acid ce set forth in SEQ ID NO: 14 (A12) wherein the N-terminal Cys is deleted or SEQ ID NO: 66. Exemplary nucleotide ces that encode SEQ ID NO: 66 include SEQ ID NO: 67. Also bed herein is an isolated tide ce that includes SEQ ID NO: 67.
A22: Described herein is an isolated polypeptide that includes the amino acid sequence set forth in SEQ ID NO: 15 (A22) wherein the N-terminal Cys is deleted or SEQ ID NO: 68. Exemplary nucleotide sequences that encode SEQ ID NO: 68 include SEQ ID NO: 69. Described herein is an isolated tide ce that includes SEQ ID NO: 69.
A62: Described herein is an isolated polypeptide having an amino acid sequence that is at least 95% identical to SEQ ID NO: 71, wherein the first 20 amino acid residues of the sequence does not contain a cysteine. Preferably, the polypeptide includes the amino acid sequence as shown at positions 1-184 of SEQ ID NO: 71. The polypeptide is ably non-lipidated and non-pyruvylated. In another embodiment, the polypeptide is immunogenic.
In another embodiment, the isolated polypeptide includes a fragment of A62.
Exemplary fragments of A62 includes any number of contiguous residues from SEQ ID NO: 70 or SEQ ID NO: 71. In one embodiment, the isolated polypeptide includes the amino acid ce at positions 158-185 of SEQ ID NO: 71. In another embodiment, the isolated polypeptide includes the amino acid sequence at positions 159-186 of SEQ ID NO: 71. In one embodiment, the polypeptide includes at least 6 contiguous amino acids from the amino acid ce at positions 185-254 of SEQ ID NO: 71.
Described herein is an isolated nucleic acid sequence encoding an isolated polypeptide having an amino acid sequence that is at least 95% identical to SEQ ID NO: 71, wherein the first 20 amino acid residues of the sequence does not contain a cysteine. Preferably, the polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 71. In one embodiment, the isolated c acid sequence includes SEQ ID NO: 72. bed herein is an ed polypeptide that includes the amino acid sequence set forth in SEQ ID NO: 70 (A62) wherein the inal Cys is deleted or SEQ ID NO: 71. Exemplary nucleotide sequences that encode SEQ ID NO: 71 include SEQ ID NO: 72. Described herein is an isolated nucleotide sequence that includes SEQ ID NO: 72.
Immunogenic Compositions In a preferred embodiment, the compositions described herein including an isolated ruvylated non-lipidated ORF2086 polypeptide are immunogenic.
Immunogenic compositions that include a protein encoded by a nucleotide sequence from Neisseria meningitidis ORF2086 are known in the art. Exemplary immunogenic compositions e those described in WO2003/063766, and US patent ation publication numbers US 57413 and US 20090202593, which are incorporated herein by reference in their entirety. Such immunogenic compositions described therein include a protein exhibiting bactericidal activity identified as ORF2086 protein, immunogenic portions thereof, and/or biological equivalents thereof. The ORF2086 protein refers to a protein encoded by open g frame 2086 of Neisseria species.
The protein may be a recombinant protein or an isolated protein from native Neisseria species. For example, Neisseria ORF2086 proteins may be isolated from bacterial strains, such as those of ria species, including strains of Neisseria meningitidis (serogroups A, B, C, D, W-135, X, Y, Z, and 29E), Neisseria gonorrhoeae, and Neisseria ica, as well as immunogenic portions and/or biological equivalents of said proteins.
The ORF2086 proteins include 2086 Subfamily A proteins and Subfamily B proteins, immunogenic portions thereof, and/or ical equivalents f. 2086 subfamily A proteins and 2086 ily B proteins are known in the art, see, for example Fletcher et al., 2004 cited above and Murphy et al., J Infect Dis. 2009 Aug 1;200(3):379-89. See also WO2003/063766, which discloses SEQ ID NOs: 260 to 278 therein as representing amino acid sequences ated with proteins of 2086 Subfamily A. In on, disclosed in WO2003/063766 are SEQ ID NOS: 279 to 299 therein as representing amino acid sequences associated with proteins of 2086 Subfamily B. /063766 is incorporated herein by reference in its entirety. The ORF2086 proteins or equivalents thereof, etc. may be lipidated or non ted.
Preferably, the Neisseria ORF2086 protein is non ted. Alternatively, the immunogenic compositions may be combinations of lipidated and non lipidated ORF2086 proteins.
In (an) one embodiment, the immunogenic ition includes an isolated protein having at least 95% amino acid sequence identity to a protein encoded by a nucleotide sequence from Neisseria ORF2086. In another embodiment, the immunogenic composition es an isolated protein having at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence identity to a protein encoded by a nucleotide sequence from Neisseria ORF2086.
In one embodiment, the immunogenic composition includes an isolated protein having at least 95% amino acid sequence identity to a ily A protein encoded by a nucleotide sequence from Neisseria ORF2086. Preferably, the immunogenic composition includes an isolated Subfamily A protein encoded by a nucleotide ce from Neisseria ORF2086. In some embodiments, the ORF2086 Subfamily A polypeptide is an A05, an A04, an A12, an A62, or an A22 variant. In some embodiments, the ORF2086 Subfamily A polypeptide is an A05, an A12, or an A22 variant.
Combination of subfamily A polypeptides: In one embodiment, the composition includes any combination of ORF2086 ily A polypeptides.
Exemplary combinations of ORF2086 Subfamily A polypeptides include, for example, A05 and A12; A05 and A22; A05 and A62; A12 and A62; A12 and A22; A22 and A62; A05, A12, and A22; A05, A12, and A62; A12, A22, and A62; and A05, A22, and A62.
Preferably, the ORF2086 Subfamily A polypeptide is non-lipidated and non-pyruvylated.
In another embodiment, the immunogenic composition es an isolated protein having at least 95% amino acid sequence ty to a Subfamily B protein encoded by a nucleotide sequence from Neisseria ORF2086. Preferably, the immunogenic ition includes an isolated Subfamily B protein encoded by a nucleotide sequence from Neisseria ORF2086. In some embodiments, the ORF2086 Subfamily B protein is a B44, a B02, a B03, a B22, a B24 or a B09 variant. In some ments, the ORF2086 Subfamily B n is a B44, a B22, or a B09 variant.
Combination of subfamily B polypeptides: In one embodiment, the composition es any combination of ORF2086 Subfamily B polypeptides.
Exemplary combinations of ORF2086 Subfamily B polypeptides include, for example, B09 and B22; B22 and B44; B44 and B09; B01 and B09; B01 and B22; B01 and B44; and B09, B22, and B44; B09 and B24; B22 and B24; B24 and B44; B01 and B24; B02 and B24; B02 and B01; B02 abd B09; B02 and B44; B01, B09, and B24; B01, B24, and In a preferred embodiment, the genic composition includes an isolated non-pyruvylated non-lipidated polypeptide having at least 95% amino acid sequence identity to a Subfamily B protein encoded by a tide sequence from Neisseria ORF2086. For example, in some embodiments, the ORF2086 Subfamily B n is sequences selected from a B44 having an amino acid sequence as shown in SEQ ID NO: 21; a B02 having an amino acid sequence as shown in SEQ ID NO: 16; a B03 having an amino acid sequence as shown in SEQ ID NO: 17; a B22 having an amino acid sequence as shown in SEQ ID NO:19; a B24 having an amino acid sequence as shown in SEQ ID NO: 20; a B01 having an amino acid sequence as shown in SEQ ID NO:58; or a B09 variant having an amino acid sequence as shown in SEQ ID NO:18, wherein the N-terminal Cys is deleted, or a ation thereof.
More preferably, the immunogenic composition includes a non-pyruvylated nonlipidated B09 polypeptide, a non-pyruvylated non-lipidated B44 ptide, or combinations thereof. In one embodiment, the composition includes a non-pyruvylated non-lipidated B09 variant having the amino acid sequence as shown in SEQ ID NO:18, wherein the N-terminal Cys is d, a non-pyruvylated non-lipidated B44 having the amino acid sequence as shown in SEQ ID NO: 21, wherein the N-terminal Cys is deleted, or a ation thereof. In another embodiment, the immunogenic composition includes a non-pyruvylated non-lipidated B09 having SEQ ID NO: 49, a ruvylated non-lipidated B44 having SEQ ID NO: 44, or a combination thereof.
Described herein is an immunogenic composition that includes an ORF2086 subfamily B polypeptide from serogroup B N. meningitidis, wherein the polypeptide is a non-pyruvylated non-lipidated B44. The B44 may include the amino acid sequence as shown in SEQ ID NO: 21, wherein the inal Cys is d or SEQ ID NO: 44. In one embodiment, the composition further es a second ORF2086 subfamily B ptide from serogroup B N. meningitidis, wherein the second polypeptide is a nonpyruvylated non-lipidated B09. The B09 may include the amino acid sequence as shown in SEQ ID NO: 18, wherein the N-terminal Cys is deleted, or SEQ ID NO: 49. In one embodiment, the immunogenic composition is a vaccine.
In another embodiment, the composition includes no more than 3 ORF2086 subfamily B polypeptides. In a further embodiment, the composition includes no more than 2 ORF2086 subfamily B polypeptides.
In a further embodiment, the composition includes at most 1, 2, or 3 species of an ORF2086 subfamily B variant. I n a further embodiment, the composition includes at most 1, 2, or 3 species of an ORF2086 subfamily A variant.
Compositions including a Subfamily B polypeptide and a Subfamily A polypeptide: In one embodiment, the composition further includes one or more ORF2086 ily A polypeptides. In a preferred ment, the composition includes an A05 subfamily A polypeptide. More preferably, the A05 subfamily A polypeptide is non-lipidated and non-pyruvylated. In another preferred embodiment, the composition includes an A62 subfamily A polypeptide. More preferably, the A62 subfamily A polypeptide is non-lipidated and ruvylated.
In yet another embodiment, the immunogenic composition includes an ed protein having at least 95% amino acid sequence identity to a Subfamily A protein encoded by a nucleotide sequence from Neisseria ORF2086, and an isolated protein having at least 95% amino acid sequence identity to a Subfamily B n encoded by a nucleotide sequence from Neisseria ORF2086.
Preferably, the immunogenic composition includes an isolated ily A protein encoded by a nucleotide sequence from ria ORF2086 and an isolated Subfamily B protein encoded by a nucleotide sequence from Neisseria ORF2086. More preferably, the immunogenic ition includes an isolated non-pyruvylated nonlipidated Subfamily A ORF2086 polypeptide and an isolated non-pyruvylated nonlipidated Subfamily B ORF2086 polypeptide. ations: Any combination of ORF2086 polypeptides are contemplated. In one embodiment, the composition includes at least one Subfamily A polypeptide in the absence of Subfamily B polypeptides. For example, the composition includes only Subfamily A polypeptides. In r embodiment, the ition includes at least one Subfamily B polypeptide in the e of Subfamily A polypeptides. For example, the ition es only Subfamily A polypeptides.
The genic composition may include any Subfamily A polypeptide or combination f. In some embodiments, the ORF2086 Subfamily A polypeptide is an A05, an A04, an A12, or an A22 variant. In another embodiment, the ORF2086 Subfamily A polypeptide includes A62. In a preferred embodiment, the ORF2086 Subfamily A polypeptide is an A05 having an amino acid sequence as shown in SEQ ID NO: 13; an A04 having an amino acid sequence as shown in SEQ ID NO: 12; an A12 having an amino acid sequence as shown in SEQ ID NO: 14; or an A22 variant having an amino acid sequence as shown in SEQ ID NO: 15, wherein the N-terminal Cys is deleted, or any combination thereof. Yet another exemplary immunogenic composition includes a combination of isolated non-pyruvylated non-lipidated A05 and A62 Subfamily A ORF2086 ptides. For example, the immunogenic composition may include a polypeptide having SEQ ID NO: 55 and a polypeptide having SEQ ID NO: 71.
A further exemplary immunogenic composition includes a combination of isolated uvylated non-lipidated A05 and A12 Subfamily A ORF2086 polypeptides. Another exemplary immunogenic composition includes a combination of isolated nonpyruvylated non-lipidated A12 and A62 Subfamily A 6 polypeptides.
The immunogenic composition may include any Subfamily B polypeptide or combination thereof. In some embodiments, the ORF2086 ily B protein is a B44, a B02, a B03, a B22, a B24 or a B09 variant. In a preferred embodiment, the ORF2086 Subfamily B protein is a B44 having the amino acid sequence as shown in SEQ ID NO: 21; a B02 having an amino acid sequence as shown in SEQ ID NO: 16; a B03 having an amino acid sequence as shown in SEQ ID NO: 17; a B22 having an amino acid sequence as shown in SEQ ID NO:19; a B24 having an amino acid sequence as shown in SEQ ID NO: 20; or a B09 variant having an amino acid sequence as shown in SEQ ID NO:18, wherein the N-terminal Cys is deleted, or a ation thereof. Yet another exemplary immunogenic composition includes a combination of isolated non-pyruvylated non-lipidated B09 and B44 Subfamily B ORF2086 polypeptides. A further exemplary immunogenic composition includes a combination of isolated non-pyruvylated non-lipidated B09 and B22 Subfamily B ORF2086 polypeptides. Another exemplary immunogenic composition includes a ation of isolated non-pyruvylated non-lipidated B22 and B44 ily B 6 ptides. An additional exemplary immunogenic composition includes a ation of isolated non-pyruvylated non-lipidated B09, B22, and B44 Subfamily B ORF2086 polypeptides.
In one embodiment, the ition includes a non-lipidated ORF2086 polypeptide in the e of a ted ORF2086 polypeptide. In another embodiment, the composition es a non-lipidated ORF2086 polypeptide and at least one lipidated ORF2086 polypeptide.
In one embodiment, the composition es a non-pyruvylated non-lipidated ORF2086 ptide in the absence of a lipidated ORF2086 polypeptide. In another embodiment, the composition includes a lipidated ORF2086 polypeptide and a nonpyruvylated pidated 6 polypeptide. For example, the ition may include a lipidated A05 ptide having SEQ ID NO: 76 and a non-pyruvylated nonlipidated A05 having SEQ ID NO: 77. Another exemplary composition includes a lipidated A05 ptide having SEQ ID NO: 76 and a non-pyruvylated non-lipidated A62 having SEQ ID NO: 71. An onal exemplary composition includes a lipidated B01 polypeptide having SEQ ID NO: 58 and a non-pyruvylated non-lipidated A62 having SEQ ID NO: 71.
Exemplary combinations: One exemplary immunogenic ition includes a combination of an isolated non-lipidated A05, B09, B22, and B44 ORF2086 polypeptides. For example, the genic composition may include a nonpyruvylated non-lipidated A05 (SEQ ID NO: 55) ily A ORF2086 polypeptide and isolated non-pyruvylated non-lipidated B09 (SEQ ID NO: 49), B22 (SEQ ID NO: 75), and B44 (SEQ ID NO: 44) Subfamily B ORF2086 polypeptides.
Another exemplary immunogenic composition includes a combination of isolated non-pyruvylated non-lipidated A05 and A12 Subfamily A 6 polypeptides and isolated non-pyruvylated non-lipidated B22 and B44 Subfamily B ORF2086 polypeptides. A further exemplary immunogenic composition includes isolated nonpyruvylated non-lipidated A05, A12, B09, and B44 polypeptides. Yet another example includes isolated non-pyruvylated non-lipidated A12, A62, B09, and B44 polypeptides.
Yet a r example includes isolated non-pyruvylated non-lipidated A05, A12, A62, B09, and B44 polypeptides. Another exemplary immunogenic composition includes isolated non-pyruvylated pidated A62 and B09 polypeptides. Another exemplary immunogenic composition includes isolated ruvylated non-lipidated A62 and B44 polypeptides. Another exemplary immunogenic composition includes isolated nonpyruvylated non-lipidated A62, B09, and B44 polypeptides. Another exemplary immunogenic composition includes isolated non-pyruvylated non-lipidated A05, A62, and B44 polypeptides. Another exemplary immunogenic composition includes isolated non-pyruvylated non-lipidated A05, A62, B09, and B44 polypeptides.
In one embodiment, the immunogenic composition includes a 1:1 ratio of a Subfamily A protein to a Subfamily B protein. In r embodiment, the immunogenic composition includes any one of the following ratios of a Subfamily A ptide to a Subfamily B polypeptide: 1:1; 1:2; 1:3; 1:4; 1:5; 1:6; 1:7; 1:8; 1:9; or 1:10. In another embodiment, the immunogenic composition includes any one of the following ratios of a Subfamily B polypeptide to a Subfamily A polypeptide: 1:1; 1:2; 1:3; 1:4; 1:5; 1:6; 1:7; 1:8; 1:9; or 1:10.
Bactericidal immune responses In one , the isolated polypeptides and compositions bed herein elicit a bactericidal immune response in a mammal against infection from any serogroup of N. meningitidis, such as a serogroup selected from oup A, B, C, E29, H, I, K, L, W- 135, X , Y and Z. In a preferred embodiment, the isolated polypeptides and compositions described herein elicit a bactericidal immune response in a mammal against infection from serogroups A, B, C, W-135, Y and/or X.
In another , the isolated polypeptides and compositions bed herein elicit a bactericidal immune response in a mammal against an ORF2086 polypeptide from serogroup B N. meningitidis. The compositions have the ability to induce bactericidal anti-meningococcal antibodies after administration to a mammal, and in red embodiments can induce antibodies that are bactericidal against strains with the respective subfamilies. Further information on bactericidal responses is given below. See, for example, Examples 6, 11, 12, and 13.
In one embodiment, the compositions elicit a bactericidal immune response against a heterologous subfamily of N. meningitidis oup B. For example, a composition including a non-lipidated ily A polypeptide may elicit a bactericidal immune response against a subfamily A variant of N. meningitidis serogroup B and/or against a subfamily B variant of N. meningitidis serogroup B. See, for example, Examples 18-19.
In a further aspect, the isolated polypeptides and compositions bed herein elicit a bactericidal immune response against at least one of serogroup A, serogroup B, oup C, serogroup W135, and/or serogroup Y strains of N. meningitidis. In a red embodiment, the compositions elicit a bactericidal immune response at least t serogroup B, serogroup C, and serogroup Y of N. meningitidis. See, for example, Example 21.
Bactericidal antibodies are an indicator of protection in humans and preclinical s serve as a surrogate, and any new immunogenic composition candidate described herein should elicit these functional antibodies.
B09: In one aspect, the isolated pidated B09 polypeptide, and immunogenic compositions thereof, elicits bactericidal antibodies against (e.g., that can bind to) an 6 polypeptide from serogroup B N. meningitidis, subfamily B. In an exemplary embodiment, the isolated non-pyruvylated non-lipidated B09 polypeptide having SEQ ID NO: 18 wherein the N-terminal Cys at position 1 is deleted or SEQ ID NO: 49, and immunogenic compositions thereof, elicits bactericidal antibodies against (e.g., that can bind to) an ORF2086 polypeptide from serogroup B N. meningitidis, subfamily A or preferably subfamily B. Preferably, the non-pyruvylated non-lipidated B09 polypeptide and immunogenic compositions thereof, elicits bactericidal antibodies against the A05 variant (SEQ ID NO: 13); B44 variant (SEQ ID NO: 21); B16 variant (SEQ ID NO: 60); B24 t (SEQ ID NO: 20); B09 variant (SEQ ID NO: 18), or a ation thereof. In an exemplary embodiment, the non-pyruvylated non-lipidated B09 polypeptide and immunogenic compositions thereof, elicits bactericidal antibodies against B44 variant (SEQ ID NO: 21); B16 variant (SEQ ID NO: 60); B24 variant (SEQ ID NO: 20); B09 variant (SEQ ID NO: 18), or a combination thereof. See, for example, Example 11, Example 12, and Example 13.
B44: In one aspect, the isolated pidated B44 polypeptide, and immunogenic compositions thereof, elicits bactericidal antibodies against (e.g., that can bind to) an ORF2086 polypeptide from serogroup B N. meningitidis, subfamily B. In another exemplary embodiment, the isolated non-pyruvulated non-lipidated B44 polypeptide having SEQ ID NO: 21 wherein the N-terminal Cys at position 1 is deleted or SEQ ID NO: 44, and immunogenic compositions thereof, elicits bactericidal dies against (e.g., that can bind to) an ORF2086 polypeptide from serogroup B N. meningitidis, ily B. Preferably, the non-pyruvylated non-lipidated B44 polypeptide and genic compositions thereof, s bactericidal antibodies t the B44 variant (SEQ ID NO: 21); B16 variant (SEQ ID NO: 60); B24 t (SEQ ID NO: 20); B09 variant (SEQ ID NO: 18), or a ation thereof. See, for example, Example 11. Additionally, the non-pyruvylated non-lipidated B44 polypeptide and immunogenic compositions thereof may also elicit icidal dies that bind to the B02 variant (SEQ ID NO: 16). See, for example, Example 12 and Example 13.
Moreover, the non-pyruvylated non-lipidated B44 polypeptide and immunogenic compositions thereof may also elicit bactericidal antibodies that bind to B03 variant (SEQ ID NO: 17) and B15 variant (SEQ ID NO: 59). See, for example, Example 6.
B22: In one aspect, the isolated non-lipidated B22 polypeptide, and genic compositions thereof, elicits bactericidal antibodies against (e.g., that can bind to) an ORF2086 ptide from serogroup B N. meningitidis, subfamily B. In a further exemplary embodiment, the isolated non-pyruvulated non-lipidated B22 polypeptide having SEQ ID NO: 19 wherein the N-terminal Cys at position 1 is deleted, and immunogenic compositions thereof, elicits bactericidal antibodies against (e.g., that can bind to) an ORF2086 polypeptide from serogroup B N. itidis, subfamily B.
Preferably, the non-pyruvylated non-lipidated B22 polypeptide elicits icidal dies against the B44 variant (SEQ ID NO: 21); B16 variant (SEQ ID NO: 60); B24 variant (SEQ ID NO: 20); B09 variant (SEQ ID NO: 18), or a combination thereof. See, for example, Example 13.
A05: In one aspect, the isolated non-lipidated A05 polypeptide, and immunogenic compositions thereof, elicits bactericidal antibodies against (e.g., that can bind to) an ORF2086 polypeptide from serogroup B N. itidis, subfamily A. In one embodiment, the ed non-pyruvylated non-lipidated A05 polypeptide having SEQ ID NO: 13 wherein the inal Cys is deleted or SEQ ID NO: 55, and immunogenic compositions thereof, elicits bactericidal dies against (e.g., that can bind to) an ORF2086 polypeptide from serogroup B N. itidis, subfamily A. In one embodiment, the isolated A05 polypeptide includes the amino acid ce SEQ ID NO: 76, wherein the cysteine at position 1 is deleted. In r embodiment, the isolated A05 polypeptide includes the amino acid sequence SEQ ID NO: 76, n the cysteine at position 1 is substituted with an amino acid that is not a Cys residue. In one embodiment, the ed A05 polypeptide includes the amino acid sequence SEQ ID NO: 77. Preferably, the non-pyruvylated non-lipidated A05 and immunogenic compositions thereof, elicits bactericidal antibodies against the A05 variant (SEQ ID NO: 13), A22 variant (SEQ ID NO: 15), A12 variant (SEQ ID NO: 14), or a combination thereof. See, for example, Example 6 and 13.
A62: In one aspect, the isolated non-lipidated A62 polypeptide, and immunogenic compositions thereof, elicits bactericidal antibodies against (e.g., that can bind to) an ORF2086 polypeptide from oup B N. meningitidis, subfamily A. In one embodiment, the isolated A62 polypeptide includes the amino acid sequence SEQ ID NO: 70, wherein the cysteine at position 1 is substituted with an amino acid that is not a Cys residue. In another embodiment, the isolated non-pyruvylated non-lipidated A62 polypeptide having SEQ ID NO: 70 wherein the N-terminal Cys is deleted or SEQ ID NO: 71, and genic compositions f, elicits bactericidal antibodies against (e.g., that can bind to) an 6 polypeptide from serogroup B N. itidis, subfamily A and/or subfamily B. For example, the non-pyruvylated non-lipidated A62 and genic compositions thereof, elicits bactericidal antibodies against the A05 variant (SEQ ID NO: 13), A12 variant (SEQ ID NO: 14), A22 variant (SEQ ID NO: 15), and A62 variant (SEQ ID NO: 70). As another example, the non-pyruvylated nonlipidated A62 and immunogenic compositions thereof, elicits bactericidal antibodies against the A29 t, B09 variant, and B24 variant. See, for example, Examples 18- 19. In another embodiment, the non-pyruvylated non-lipidated A62 and immunogenic compositions thereof, elicits bactericidal antibodies against the B16 variant.
A12: In one embodiment, the isolated non-pyruvylated non-lipidated A12 polypeptide having SEQ ID NO: 14 wherein the N-terminal Cys is deleted or SEQ ID NO: 66, and immunogenic compositions thereof, elicits bactericidal antibodies against an ORF2086 polypeptide from serogroup B N. meningitidis, ily A and/or ily B. Preferably, the non -pyruvylated non-lipidated A12 and immunogenic compositions thereof, elicits icidal antibodies against the A05 variant (SEQ ID NO: 13), A22 variant (SEQ ID NO: 15), A12 variant (SEQ ID NO: 14), A62 variant (SEQ ID NO: 70), A29 variant, B09 variant. See, for exa mple, Examples 18-19.
In one embodiment, the isolated non-pyruvylated non-lipidated A22 polypeptide having SEQ ID NO: 15 wherein the N-terminal Cys is deleted or SEQ ID NO: 68, and immunogenic compositions thereof, elicits bactericidal antibodies against (e.g., that can bind to) an ORF2086 polypeptide from serogroup B N. meningitidis, subfamily A and/or subfamily B. ably, the non -pyruvylated non-lipidated A22 and immunogenic compositions thereof, elicits bactericidal antibodies against the A05 variant (SEQ ID NO: 13), A22 t (SEQ ID NO: 15), A62 variant (SEQ ID NO: 70), A29 t. See, for e, Examples 18-19.
Method of ing bactericidal antibodies Described herein is a method of eliciting bactericidal antibodies specific to serogroup A N. meningitidis in a mammal. bed herein is a method of eliciting bactericidal antibodies specific to serogroup C N. itidis in a mammal. Described herein is a method of eliciting bactericidal antibodies specific to serogroup W135 N. meningitidis in a mammal. Described herein is a method of eliciting bactericidal antibodies specific to serogroup X N. meningitidis in a mammal. Described herein is a method of eliciting bactericidal antibodies specific to oup Y N. meningitidis in a mammal. Described herein is a method of eliciting bactericidal antibodies specific to serogroups A, B, C, W-135, X and/or Y N. meningitidis in a . bed herein is a method of ing bactericidal dies specific to serogroup B N. meningitidis in a mammal. In an exemplary embodiment, the method includes eliciting icidal antibodies specific to an ORF2086 subfamily B serogroup B N. meningitidis, an ORF2086 ily A serogroup B N. meningitidis, or a combination thereof.
The method includes administering to the mammal an effective amount of an isolated non-pyruvylated non-lipidated 2086 polypeptide or immunogenic composition thereof, as described above. See, for example, Examples 18-19, and 22.
In a preferred embodiment, the method includes ing bactericidal antibodies specific to an ORF2086 subfamily B serogroup B N. meningitidis. The isolated polypeptide or immunogenic composition includes a non-pyruvylated non-lipidated B44 polypeptide. In another preferred embodiment, the composition further includes a nonpyruvylated non-lipidated B09 polypeptide. In an exemplary embodiment, the isolated polypeptide or immunogenic composition includes SEQ ID NO: 49, SEQ ID NO: 44, or a combination thereof. In another exemplary embodiment, the isolated polypeptide or immunogenic composition includes SEQ ID NO: 18, wherein the N-terminal Cys at position 1 is deleted, SEQ ID NO: 21, wherein the N-terminal Cys at position 1 is deleted, or a ation thereof, In yet another exemplary embodiment, the ed polypeptide or immunogenic composition includes SEQ ID NO: 19, n the N- terminal Cys at position 1 is deleted. In one embodiment, the immunogenic composition for ing icidal antibodies specific to an ORF2086 subfamily B serogroup B N. meningitidis includes at least one of a non- pyruvylated non-lipidated A05, A12, and A62 polypeptide. See, for e, Example 19.
In a preferred ment, the method includes eliciting bactericidal antibodies specific to an ORF2086 subfamily A serogroup B N. meningitidis. The isolated polypeptide or immunogenic composition includes a non-pyruvylated non-lipidated A05 polypeptide. In a preferred embodiment, the isolated polypeptide or immunogenic composition includes SEQ ID NO: 13, wherein the N-terminal Cys at on 1 is deleted. In another preferred embodiment, the ition r includes a nonpyruvylated non-lipidated B44 polypeptide. See, for example, Example 6 and 13. In an exemplary embodiment, the isolated polypeptide or immunogenic composition includes SEQ ID NO: 55, SEQ ID NO: 44, or a combination thereof. In a preferred embodiment, the isolated polypeptide or genic composition includes SEQ ID NO: 13, wherein the N-terminal Cys at position 1 is deleted, SEQ ID NO: 21, wherein the inal Cys at position 1 is deleted, or a combination thereof. In another exemplary ment, the isolated polypeptide or immunogenic composition es SEQ ID NO: 77 (A05), SEQ ID NO: 44 (B44), or a combination thereof. In one ment, the immunogenic composition for eliciting bactericidal antibodies specific to an ORF2086 subfamily A serogroup B N. meningitidis includes at least one of a non- pyruvylated non-lipidated A05, A12, and A62 polypeptide. See, for example, e s 18-19.
When an exemplary immunogenic composition including at least two nonpyruvylated non-lipidated ORF2086 ptides as bed above was administered to mammals, the inventors surprisingly discovered that a istic bactericidal immune response may be elicited against serogroup B of ria meningitidis, as compared to an immunogenic composition including one respective non-pyruvylated non-lipidated ORF2086 polypeptide. See, for example, Example 19. Accordingly, in one embodiment, the genic composition includes at least a first non-pyruvylated non-lipidated ORF2086 polypeptide that acts synergistically with at least a second pyruvylated non-lipidated ORF2086 polypeptide to elicit an immune response against serogroup B of Neisseria meningitidis.
Described herein is a method of eliciting bactericidal antibodies specific to serogroup C of N. itidis in a mammal. The method includes administering to the mammal an effective amount of an isolated non-pyruvylated non-lipidated 2086 polypeptide from N. meningitidis serogroup B or an immunogenic composition thereof, as described above. See, for example, Example 22. In one embodiment, the polypeptide es the amino acid sequence set forth in SEQ ID NO: 71 or the amino acid ce selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, wherein the cysteine at position 1 is deleted. In one embodiment, the polypeptide includes the amino acid sequence set forth in SEQ ID NO: 71 or the amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, wherein the cysteine at position 1 is substituted with an amino acid that is not a Cys residue. In another embodiment, the immunogenic ition further includes at least one conjugate selected from: a) a conjugate of a capsular ride of Neisseria itidis serogroup A, b) a conjugate of a capsular saccharide of Neisseria meningitidis serogroup C, c) a conjugate of a capsular saccharide of Neisseria meningitidis oup W135, and d) a conjugate of a capsular saccharide of Neisseria meningitidis serogroup Y. An exemplary immunogenic composition includes at least an isolated non-pyruvylated non-lipidated A62 polypeptide and a) a conjugate of a capsular saccharide of Neisseria meningitidis serogroup A, b) a conjugate of a capsular saccharide of Neisseria meningitidis serogroup C, c) a conjugate of a capsular saccharide of Neisseria meningitidis serogroup W135, and d) a conjugate of a capsular saccharide of Neisseria meningitidis serogroup Y.
Described herein is a method of eliciting bactericidal antibodies specific to serogroup Y of N. itidis in a . The method includes administering to the mammal an effective amount of an isolated non-pyruvylated non-lipidated 2086 polypeptide from N. meningitidis oup B or an immunogenic composition thereof, as described above. See, for e, Example 22. In one ment, the polypeptide includes the amino acid sequence set forth in SEQ ID NO: 71 or the amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, wherein the cysteine at position 1 is deleted. In one embodiment, the polypeptide includes the amino acid sequence set forth in SEQ ID NO: 71 or the amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, wherein the ne at on 1 is substituted with an amino acid that is not a Cys residue. In another embodiment, the immunogenic composition further includes at least one conjugate ed from: a) a conjugate of a capsular saccharide of Neisseria meningitidis serogroup A, b) a conjugate of a capsular saccharide of Neisseria meningitidis serogroup C, c) a conjugate of a capsular ride of Neisseria meningitidis serogroup W135, and d) a conjugate of a capsular saccharide of Neisseria meningitidis serogroup Y.
Described herein is a method of eliciting bactericidal antibodies specific to serogroup X of N. meningitidis in a mammal. The method includes administering to the mammal an ive amount of an isolated non-pyruvylated pidated 2086 polypeptide from N. meningitidis serogroup B or an immunogenic composition thereof, as described above. See, for example, Example 22. In one embodiment, the ptide includes the amino acid sequence set forth in SEQ ID NO: 71 or the amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, wherein the cysteine at position 1 is d. In one embodiment, the polypeptide includes the amino acid ce set forth in SEQ ID NO: 71 or the amino acid sequence selected from the group ting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, wherein the cysteine at position 1 is substituted with an amino acid that is not a Cys residue. In another embodiment, the immunogenic ition further includes at least one conjugate selected from: a) a conjugate of a capsular ride of Neisseria meningitidis oup A, b) a conjugate of a capsular saccharide of Neisseria meningitidis serogroup C, c) a conjugate of a capsular saccharide of Neisseria meningitidis serogroup W135, and d) a conjugate of a capsular saccharide of Neisseria meningitidis serogroup Y.
When an exemplary immunogenic composition including four non-pyruvylated non-lipidated ORF2086 polypeptides and a conjugate of a capsular saccharide of each of Neisseria meningitidis serogroups A, C, W135, and Y as described above was administered to mammals, the inventors surprisingly discovered that a synergistic bactericidal immune response may be ed at least against serogroups B, C, and Y of ria itidis, as compared to an genic composition including the ORF2086 polypeptides wherein conjugates of a capsular saccharide are absent, and as compared to an genic composition including a conjugate of a capsular saccharide of each of Neisseria meningitidis serogroups A, C, W135, and Y wherein an ORF2086 polypeptide is absent. See, for e, Example 22. Accordingly, in one embodiment, the immunogenic composition includes at least one non-pyruvylated non- lipidated ORF2086 polypeptide that acts synergistically with at least one ate of a capsular ride of Neisseria meningitidis serogroup A, C, W135, and Y to elicit an immune response against ria meningitidis. The immune response elicited may be against at least one of serogroups B, C, and Y of Neisseria meningitidis.The immunogenic composition may include a protein encoded by a nucleotide ce from Neisseria ORF2086, polynucleotides, or equivalents thereof as the sole active immunogen in the immunogenic composition. Alternatively, the immunogenic ition may further include active immunogens, including other Neisseria sp. immunogenic polypeptides, or immunologically-active ns of one or more other microbial pathogens (e.g. virus, prion, bacterium, or fungus, t limitation) or capsular polysaccharide. The compositions may comprise one or more desired proteins, fragments or pharmaceutical compounds as desired for a chosen indication.
Any multi-antigen or multi-valent immunogenic composition is contemplated by the present invention. For example, the immunogenic composition may e combinations of two or more ORF2086 proteins, a combination of ORF2086 protein with one or more Por A proteins, a combination of ORF2086 protein with meningococcus serogroup A, C, Y and W135 polysaccharides and/or polysaccharide conjugates, a combination of ORF2086 protein with meningococcus and pneumococcus combinations, or a combination of any of the foregoing in a form suitable for a d administration, e.g., for l delivery. Persons of skill in the art would be readily able to formulate such multi-antigen or multi-valent immunologic compositions.
Described herein is an immunogenic ition ing an isolated nonlipidated , non-pyruvylated ORF2086 polypeptide from Neisseria meningitidis serogroup B, and at least one conjugate selected from: a) a conjugate of a capsular saccharide of Neisseria meningitidis serogroup A, b) a conjugate of a ar saccharide of Neisseria itidis serogroup C, c) a conjugate of a capsular saccharide of Neisseria meningitidis serogroup W135, and d) a ate of a capsular saccharide of Neisseria meningitidis serogroup Y.
In one embodiment, the immunogenic ition includes an ed idated , non-pyruvylated ORF2086 polypeptide from ria meningitidis serogroup B, and at least two of the ates. In another embodiment, the composition includes at least three of the conjugates. For example, the compositions may include saccharides from: serogroups A and C; oups A and W135; serogroups A and Y; serogroups C and W135; serogroups W135 and Y; serogroups A, C, and W135; oups A, C, and Y; serogroups A, W135, and Y; serogroups C and W135, and Y.
Compositions including at least one serogroup C saccharide are preferred (e.g., C and In yet another embodiment, the immunogenic composition includes an isolated non-lipidated, non-pyruvylated ORF2086 polypeptide from Neisseria meningitidis serogroup B, and four conjugates, e.g., a conjugate of a capsular saccharide of Neisseria meningitidis serogroup A; a conjugate of a capsular saccharide of Neisseria meningitidis serogroup C; a conjugate of a capsular saccharide of Neisseria meningitidis serogroup W135; and a conjugate of a capsular saccharide of Neisseria meningitidis serogroup Y.
In a preferred ment, the conjugate is a conjugate of the ar saccharide and a carrier protein. Suitable carrier proteins are known in the art.
Preferably, the carrier protein is a bacterial toxin, such as a diphtheria or tetanus toxin, or s or mutants thereof. Most preferably, the carrier protein is CRM197. For example, in one embodiment, the composition es at least one conjugate selected from (a) a conjugate of (i) the capsular saccharide of serogroup A N. itidis and (ii) CRM197; (b) a conjugate of (i) the ar saccharide of serogroup C N. meningitidis and (ii) CRM197; (c) a conjugate of (i) the capsular saccharide of serogroup W135 N. itidis and (ii) CRM197; and (d) a conjugate of (i) the capsular saccharide of serogroup Y N. meningitidis and (ii) CRM197.
The capsular saccharides of serogroups A, C, W135, and Y are characterized and known in the art. For example, the ar saccharide of serogroup A meningococcus is a homopolymer of (α 1→6)-linked N-acetyl-D-mannosamine phosphate, with partial O-acetylation in the C3 and C4 positions. Acetylation at the C-3 position can be 70-95%. Conditions used to purify the saccharide can result in de-O- acetylation (e.g. under basic conditions), but it is useful to retain OAc at this C-3 position. In some embodiments, at least 50% (e.g. at least 60%, 70%, 80%, 90%, 95% or more) of the mannosamine residues in a serogroup A saccharides are O-acetylated at the C-3 position. Acetyl groups can be replaced with blocking groups to prevent hydrolysis, and such modified saccharides are still serogroup A saccharides within the meaning of the invention.
The serogroup C capsular ride is a homopolymer of (α 2→9)-linked sialic acid (N-acetyl neuraminic acid). Most serogroup C strains have O-acetyl groups at C-7 and/or C-8 of the sialic acid residues, but some al isolates lack these O-acetyl groups.
The serogroup W135 saccharide is a polymer of sialic acid-galactose disaccharide units. Like the serogroup C saccharide, it has le O-acetylation, but at sialic acid 7 and 9 positions. The ure is written as: →4)-D-NeupNAc(7/9OAc)-α- (2→6)-D-Gal-α-(1→.
The serogroup Y saccharide is similar to the serogroup W135 saccharide, except that the haride-repeating unit includes glucose instead of galactose. The serogroup Y ure is written as: →4)-D-NeupNAc(7/9OAc)-α-(2→6)-D-Glc-α-(1→.
Like serogroup W135, it has variable O-acetylation at sialic acid 7 and 9 ons.
The saccharides used according to the invention may be O-acetylated as described above, e.g., with the same O-acetylation pattern as seen in native capsular saccharides, or they may be partially or totally de-O-acetylated at one or more positions of the saccharide rings, or they may be hyper-O- acetylated relative to the native capsular saccharides.
In one embodiment, immunogenic composition includes an isolated nonlipidated , non-pyruvylated ORF2086 polypeptide from Neisseria meningitidis serogroup B, and at least one ate selected from: a) a conjugate of a capsular saccharide of ria meningitidis serogroup A, b) a conjugate of a capsular saccharide of Neisseria meningitidis serogroup C, c) a conjugate of a capsular saccharide of Neisseria meningitidis serogroup W135, and d) a conjugate of a capsular saccharide of Neisseria itidis serogroup Y, wherein the non-lipidated, non-pyruvylated ORF2086 ptide includes at least one of the following: B44, B09, A05, B22, A12, A22, A62, B24, B16, B15, and B03. In one embodiment, the polypeptide includes the amino acid sequence selected from the group consisting of SEQ ID NO: 44, SEQ ID NO: 49, SEQ ID NO: 55, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 71, and SEQ ID NO: 75. In another embodiment, the polypeptide es the amino acid sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 20, wherein the cysteine at position 1 is deleted. In another embodiment, the polypeptide es the amino acid sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 20, wherein the cysteine at position 1 is tuted with an amino acid that is not a Cys residue.
The present invention also contemplates multi-immunization regimens wherein any composition useful against a pathogen may be combined therein or therewith the compositions of the present invention. For example, without tion, a patient may be administered the immunogenic composition of the present invention and another immununological composition for immunizing against human papillomavirus virus (HPV), such as the HPV vaccine GARDASIL®, as part of a multi-immunization regimen.
Persons of skill in the art would be readily able to select immunogenic compositions for use in conjunction with the immunogenic compositions of the present invention for the purposes of developing and implementing immunization ns.
The ORF2086 ptides, fragments and equivalents can be used as part of a conjugate immunogenic composition; wherein one or more proteins or polypeptides are conjugated to a carrier in order to generate a composition that has genic properties against several serotypes, or serotypes of N. meningitidis, specifically meningococcus serogroups specifically serogroup B, and/or against several diseases. atively, one of the ORF2086 ptides can be used as a carrier protein for other immunogenic polypeptides. Formulation of such immunogenic compositions is well known to persons skilled in this field.
Immunogenic compositions of the invention ably include a pharmaceutically acceptable ent, diluents, and/or carrier. Suitable ceutically acceptable excipients, carriers and/or diluents include any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous ons, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Suitable pharmaceutically acceptable excipients, diluents, and/or carriers include, for example, one or more of water, , phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof.
Pharmaceutically acceptable excipients, diluents, and/or carriers may further include minor amounts of ary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or iveness of the antibody.
The preparation and use of pharmaceutically acceptable excipients, diluents, and/or carriers is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the immunogenic compositions of the present invention is contemplated.
Immunogenic compositions can be administered parenterally, e.g., by injection, either subcutaneously or intramuscularly, as well as orally or intranasally. Methods for intramuscular immunization are described by Wolff et al. Biotechniques;11(4):474-85, (1991). and by Sedegah et al. PNAS Vol. 91, pp. 9866-9870, (1994). Other modes of administration employ oral formulations, pulmonary ations, suppositories, and transdermal ations, for example, without limitation. Oral formulations, for example, e such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, ose, magnesium carbonate, and the like, without limitation. Preferably, the immunogenic composition is administered intramuscularly.
The immunogenic compositions of the present ion can further se one or more onal "immunomodulators", which are agents that perturb or alter the immune system, such that either up-regulation or egulation of humoral and/or cell-mediated immunity is ed. In one particular embodiment, up-regulation of the l and/or cell-mediated arms of the immune system is preferred. es of certain immunomodulators include, for example, an adjuvant or cytokine, or ISCOMATRIX (CSL Limited, lle, Australia), described in U.S. Patent No. ,254,339 among others.
Non-limiting examples of adjuvants that can be used in the vaccine described herein include the RIBI adjuvant system (Ribi Inc., Hamilton, Mont.), alum, l gels such as aluminum hydroxide gel, oil-in-water emulsions, water-in-oil emulsions such as, e.g., Freund's te and incomplete adjuvants, Block copolymer (CytRx, Atlanta Ga.), QS-21 (Cambridge Biotech Inc., dge Mass.), SAF-M (Chiron, Emeryville Calif.), AMPHIGEN® adjuvant, saponin, Quil A or other saponin fraction, monophosphoryl lipid A, and Avridine lipid-amine adjuvant. Non-limiting examples of oil-in-water emulsions useful in the vaccine described herein include modified SEAM62 and SEAM 1/2 formulations. Modified SEAM62 is an oil-in-water emulsion containing % (v/v) squalene (Sigma), 1% (v/v) SPAN® 85 detergent (ICI Surfactants), 0.7% (v/v) polysorbate ® 80 detergent (ICI Surfactants), 2.5% (v/v) ethanol, 200 μg/ml Quil A, 100 μg/ml cholesterol, and 0.5% (v/v) lecithin. Modified SEAM 1/2 is an oil-in-water emulsion comprising 5% (v/v) squalene, 1% (v/v) SPAN® 85 detergent, 0.7% (v/v) polysorbate 80 detergent, 2.5% (v/v) ethanol, 100 μg/ml Quil A, and 50 μg/ml cholesterol.
Other "immunomodulators" that can be ed in the vaccine include, e.g., one or more interleukins, interferons, or other known cytokines or chemokines. In one embodiment, the adjuvant may be a cyclodextrin derivative or a polyanionic polymer, such as those described in U.S. patent numbers 6,165,995 and 310, respectively.
It is to be understood that the immunomodulator and/or adjuvant to be used will depend on the subject to which the vaccine or immunogenic composition will be administered, the route of injection and the number of injections to be given.
In some ments, the adjuvant is saponin. In some embodiments, the n concentration is between 1 μg/ml and 250 μg/ml; between 5 μg/ml and 150 μg/ml; or between 10 μg/ml and 100 μg/ml. In some ments, the saponin concentration is about 1 μg/ml; about 5 μg/ml; about 10 μg/ml; about 20 μg/ml; about μg/ml; about 40 μg/ml; about 50 μg/ml; about 60 μg/ml; about 70 μg/ml; about 80 μg/ml; about 90 μg/ml; about 100 μg/ml; about 110 μg/ml; about 120 μg/ml; about 130 μg/ml; about 140 μg/ml; about 150 μg/ml; about 160 μg/ml; about 170 μg/ml; about 180 μg/ml; about 190 μg/ml; about 200 μg/ml; about 210 μg/ml; about 220 μg/ml; about 230 μg/ml; about 240 μg/ml; or about 250 μg/ml.
In certain preferred embodiments, the proteins described herein are used in an immunogenic composition for oral administration which includes a mucosal adjuvant and used for the treatment or prevention of N. meningitidis infection in a human host.
The mucosal nt can be a cholera toxin; however, preferably, mucosal adjuvants other than cholera toxin which may be used in ance with the present ion include xic derivatives of a cholera holotoxin, wherein the A subunit is mutagenized, chemically modified cholera toxin, or related proteins produced by modification of the cholera toxin amino acid sequence. For a specific cholera toxin which may be particularly useful in preparing immunogenic compositions of this invention, see the mutant cholera holotoxin E29H, as disclosed in hed ational Application WO 00/18434, which is hereby incorporated herein by reference in its entirety. These may be added to, or conjugated with, the polypeptides of this invention. The same techniques can be applied to other molecules with mucosal nt or ry properties such as Escherichia coli heat labile toxin (LT).
Other compounds with mucosal adjuvant or delivery activity may be used such as bile; polycations such as DEAE-dextran and polyornithine; detergents such as sodium dodecyl benzene sulphate; lipid-conjugated materials; antibiotics such as streptomycin; vitamin A; and other compounds that alter the structural or functional integrity of mucosal surfaces. Other mucosally active compounds include derivatives of microbial structures such as MDP; acridine and cimetidine. STIMULON™ QS-21, MPL, and IL-12, as described above, may also be used.
The immunogenic compositions of this invention may be delivered in the form of ISCOMS (immune stimulating complexes), ISCOMS containing CTB, liposomes or encapsulated in compounds such as acrylates or poly(DL-lactide-co- glycoside) to form microspheres of a size suited to adsorption. The proteins described herein may also be incorporated into oily emulsions.
An amount (i.e., dose) of immunogenic composition that is administered to the patient can be determined in accordance with standard ques known to those of ordinary skill in the art, taking into consideration such s as the particular antigen, the adjuvant (if present), the age, sex, weight, species, condition of the particular patient, and the route of administration.
For example, a dosage for an adolescent human patient may include at least 0.1µg, 1 µg, 10 µg, or 50 µg of a Neisseria ORF2086 protein, and at most 80 µg, 100 µg, 150 µg, or 200 µg of a Neisseria ORF2086 n. Any minimum value and any maximum value may be combined to define a le range.
Adjuvants Immunogenic compositions as described herein also comprise, in certain embodiments, one or more nts. An adjuvant is a substance that enhances the immune response when administered together with an immunogen or antigen. A number of cytokines or kines have been shown to have immune modulating activity, and thus are useful as adjuvants, including, but not limited to, the interleukins 1-α, 1-β, 2, 4, 5, 6, 7, 8, 10, 12 (see, e.g., U.S. Patent No. 5,723,127), 13, 14, 15, 16, 17 and 18 (and its mutant ; the interferons-α, β and γ; granulocyte-macrophage colony ating factor (GM-CSF) (see, e.g., U.S. Patent No. 5,078,996 and ATCC Accession Number 39900); macrophage colony ating factor (M-CSF); granulocyte colony stimulating factor (G-CSF); and the tumor is factors α and β.
Still other adjuvants that are useful with the genic compositions described herein include chemokines, ing without limitation, MCP-1, MIP-1α, MIP-1β, and RANTES; adhesion molecules, such as a selectin, e.g., L-selectin, ctin and E-selectin; mucin-like molecules, e.g., CD34, GlyCAM-1 and MadCAM-1; a member of the integrin family such as LFA-1, VLA-1, Mac-1 and p150.95; a member of the globulin superfamily such as PECAM, ICAMs, e.g., ICAM-1, ICAM-2 and , CD2 and LFA-3; co-stimulatory les such as B7-1, B7-2,CD40 and CD40L; growth factors including vascular growth factor, nerve growth factor, fibroblast growth factor, epidermal growth factor, PDGF, BL-1, and vascular endothelial growth factor; receptor molecules including Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, and DR6; and Caspase (ICE).
Other exemplary adjuvants include, but are not limited to um hydroxide; aluminum phosphate; STIMULON™ QS-21 (Aquila Biopharmaceuticals, Inc., Framingham, Mass.); MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, Mont.), 529 (an amino alkyl glucosamine phosphate compound, Corixa, Hamilton, Mont.), IL-12 (Genetics Institute, Cambridge, Mass.); GM-CSF (Immunex Corp., Seattle, Wash.); yl-muramyl-L-theronyl-D-isoglutamine (thr-MDP); N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP); ylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine(1′-2 ′-dipalmitoyl-sn-glycerohydroxyphos-phoryloxy-ethylamin e) (CGP 19835A, referred to as MTP-PE); and a toxin. In certain preferred embodiments, the adjuvant is QS-21.
Additional exemplary adjuvants include non-toxic derivatives of cholera toxin, including its A subunit, and/or conjugates or genetically engineered fusions of the N. meningitidis polypeptide with cholera toxin or its B subunit (“CTB”), procholeragenoid, fungal polysaccharides, including schizophyllan, l ide, muramyl dipeptide (“MDP”) derivatives, phorbol esters, the heat labile toxin of E. coli , block polymers or saponins.
Aluminum phosphate has been used as the adjuvant in a phase 1 clinical trial to a concentration 0.125 mg/dose, much lower than the limit of 0.85 mg/ dose specified by the US Code of Federal Regulations [610.15(a)]. Aluminum-containing adjuvants are widely used in humans to potentiate the immune response of antigens when administered intramuscularly or subcutaneously. In some embodiments, the concentration of aluminum in the immunogenic composition is between 0.125 μg/ml and 0.5 μg/ml; between 0.20 μg/ml and 0.40 μg/ml; or between 0.20 μg/ml and 0.30 μg/ml.
In some embodiments, the concentration of aluminum in the immunogenic composition is about 0.125 μg/ml; about 0.15 μg/ml; about 0.175 μg/ml; about 0.20 μg/ml; about 0.225 μg/ml; about 0.25 μg/ml; about 0.275 μg/ml; about 0.30 μg/ml; about 0.325 μg/ml; about 0.35 μg/ml; about 0.375 μg/ml; about 0.40 μg/ml; about 0.425 μg/ml; about 0.45 μg/ml; about 0.475 μg/ml; or about 0.50 μg/ml.
In a preferred ment, the concentration of aluminum in the genic ition is between 0.125 mg/ml and 0.5 mg/ml; between 0.20 mg/ml and 0.40 mg/ml; or n 0.20 mg/ml and 0.30 mg/ml. In some embodiments, the concentration of aluminum in the immunogenic composition is about 0.125 mg/ml; about 0.15 mg/ml; about 0.175 mg/ml; about 0.20 mg/ml; about 0.225 mg/ml; about 0.25 mg/ml; about 0.275 mg/ml; about 0.30 mg/ml; about 0.325 mg/ml; about 0.35 mg/ml; about 0.375 mg/ml; about 0.40 mg/ml; about 0.425 mg/ml; about 0.45 mg/ml; about 0.475 mg/ml; or about 0.50 mg/ml.
Suitable adjuvants used to enhance an immune response further include, without limitation, MPL™ (3-O-deacylated monophosphoryl lipid A, Corixa, Hamilton, MT), which is described in U.S. Patent No. 4,912,094. Also suitable for use as nts are synthetic lipid A analogs or aminoalkyl glucosamine phosphate compounds (AGP), or tives or analogs thereof, which are ble from Corixa (Hamilton, MT), and which are described in United States Patent No. 6,113,918. One such AGP is 2-[(R)Tetradecanoyloxytetradecanoylamino] ethyl 2-DeoxyO-phosphonoO-[(R)tetradecanoyoxytetradecanoyl ][(R)tetradecanoyloxytetradecanoyl-amino]-b-D-glucopyranoside, which is also known as 529 (formerly known as RC529). This 529 adjuvant is formulated as an aqueous form (AF) or as a stable emulsion (SE).
Still other adjuvants include muramyl peptides, such as N-acetyl-muramyl-L-threonyl-D-isoglutamine DP), N-acetyl-normuramyl-L-alanine(1'-2' dipalmitoyl-sn-glycerohydroxyphosphoryloxy )-ethylamine (MTP-PE); oil-in-water emulsions, such as MF59 (U.S. Patent No. 884) (containing 5% Squalene, 0.5% polysorbate 80, and 0.5% SPAN 85 (optionally containing various amounts of MTP-PE) formulated into submicron les using a microfluidizer such as Model 110Y microfluidizer fluidics, , MA)), and SAF (containing 10% Squalene, 0.4% polysorbate 80, 5% IC-blocked polymer L121, and thr-MDP, either microfluidized into a submicron emulsion or vortexed to te a larger particle size emulsion); incomplete Freund's adjuvant (IFA); aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate; AMPHIGEN; Avridine; qualene; D-lactide-polylactide/glycoside; PLURONIC polyols; killed Bordetella; saponins, such as Stimulon™ QS-21 (Antigenics, Framingham, MA.), described in U.S. Patent No. 5,057,540, ISCOMATRIX (CSL d, Parkville, Australia), described in U.S. Patent No. 5,254,339, and immunostimulating complexes (ISCOMATRIX); Mycobacterium tuberculosis; bacterial lipopolysaccharides; synthetic polynucleotides such as oligonucleotides containing a CpG motif (e.g., U.S. Patent No. 6,207,646); IC-31 cell AG, Vienna, Austria), bed in European Patent Nos. 1,296,713 and 1,326,634; a sis toxin (PT) or mutant thereof, a cholera toxin or mutant thereof (e.g., U.S. Patent Nos. 7,285,281, 7,332,174, 7,361,355 and 7,384,640); or an E. coli heat-labile toxin (LT) or mutant thereof, particularly LT-K63, LT-R72 (e.g., U.S. Patent Nos. 6,149,919, 7,115,730 and 7,291,588).
Methods of Producing Non-Lipidated P2086 Antigens Described herein is a method of producing a non-pyruvylated non-lipidated ORF2086 polypeptide. The method includes expressing a nucleotide sequence encoding an ORF2086 polypeptide wherein the N-terminal ne is deleted as compared to the corresponding wild-type sequence, and n the nucleotide sequence is operatively linked to an expression system that is capable of being expressed in a bacterial cell. Exemplary polypeptides ed by the method include any polypeptide bed herein. For example, preferably, the polypeptide has the amino acid sequence set forth in SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 58; SEQ ID NO: 70, wherein the cysteine at position 1 is deleted, as compared to the corresponding wild-type sequence. In another preferred embodiment, the polypeptide has the amino acid sequence set forth in SEQ ID NO: 76, wherein the cysteine at position 1 is deleted. Additional exemplary polypeptides include a polypeptide having the amino acid sequences selected from SEQ ID NO: 44, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 71, and SEQ ID NO: 75. An additional exemplary polypeptide includes a polypeptide having the amino acid ce SEQ ID NO: 77. Further examples include SEQ ID NO: 80 (B24) and SEQ ID NO: 81 (B24). The method further includes purifying the polypeptide.
Described herein is a method for producing soluble non-lipidated P2086 antigens comprising the steps of cloning the ORF2086 t nucleic acid sequence into an E. coli expression vector without a lipidation control ce, transforming E. coli bacteria with the ORF2086 expression vector, inducing expression and isolating the expressed P2086 protein. In some embodiments, expression is induced with IPTG.
In some embodiments, the codon for the N-terminal Cys of the ORF2086 variant is deleted. Examples of such codons include TGC. In some ments, the codon for the N-terminal Cys of the ORF2086 variant is mutated by point mutagenesis to generate an Ala, Gly, or Val codon. In some embodiments, Ser and Gly codons are added to the N-terminal tail of the ORF2086 variant to extend the Gly/Ser stalk immediately ream of the N-terminal Cys. In some embodiments, the total number of Gly and Ser residues within the Gly/Ser stalk is at least 7, 8, 9, 10, 11, or 12.
In some embodiments, the codon for the N-terminal Cys is deleted. In some embodiments, the N-terminal 7, 8, 9, 10, 11, or 12 residues are either Gly or Ser.
In some embodiments, the codons of the N-terminal tail of the non-lipidated 6 t are optimized by point mutagenesis. In some embodiments, the N-terminal tail of the non-lipidated ORF2086 variant is optimized to match the N-terminal tail of the B09 variant. In some embodiments, the codons of the N-terminal tail of the ORF2086 variant are optimized by point mutagenesis such that the codon ng the fifth amino acid of the ORF2086 variant is 100% cal to nucleotides 13-15 of SEQ ID NO: 8 and the codon ng the thirteenth amino acid of the ORF2086 variant is 100% cal to nucleotides 37-39 of SEQ ID NO: 8. In some embodiments, the inal tail of the non-lipidated ORF2086 variant is optimized such that the 5’ 45 nucleic acids are 100% identical to nucleic acids 1-45 of SEQ ID NO: 8. In some embodiments, the N-terminal tail of the non-lipidated ORF2086 variant is optimized such that the 5’ 42 nucleic acids are 100% cal to nucleic acids 4-45 of SEQ ID NO: 8. In some embodiments, the N-terminal tail of the non-lipidated ORF2086 variant is optimized such that the 5’ 39 nucleic acids are 100% identical to nucleic acids 4-42 of SEQ ID NO: 8. In some embodiments, the N-terminal tail of the non-lipidated P2086 variant comprises at least one amino acid substitution compared to amino acids 1-15 of SEQ ID NO: 18. In some ments, the inal tail of the non-lipidated P2086 variant comprises two amino acid tutions compared to amino acids 1-15 of SEQ ID NO: 18. In some embodiments, the inal tail of the non-lipidated P2086 t comprises at least one amino acid substitution compared to amino acids 2-15 of SEQ ID NO: 18. In some embodiments, the N-terminal tail of the non-lipidated P2086 variant comprises two amino acid substitutions compared to amino acids 2-15 of SEQ ID NO: 18. In some embodiments, the amino acid tutions are conservative amino acid substitutions.
In some embodiments, the codons of the non-lipidated variant have been optimized for increased expression. Codon optimization is known in the art. See, e.g., Sastalla et al, Applied and Environmental Microbiology, vol. 75(7): 2099-2110 (2009) and Coleman et al, Science, vol. 320: 1784 (2008). In some embodiments, codon optimization includes matching the codon utilization of an amino acid sequence with the codon frequency of the host organism chosen while including and/or excluding specific DNA sequences. In some embodiments, codon optimization further includes minimizing the ponding secondary mRNA ure to reduce translational impediments. In some embodiments, the N-terminal tail has been codon optimized to comprise any one of SEQ ID NO: 28, 30, 32, and 34. In some embodiments, the Gly/Ser stalk has been codon optimized to comprise any one of SEQ ID NO: 28, 30, 32, and 34.
In order that this invention may be better tood, the following examples are set forth. The examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention.
Immunogenic Composition Formulations In certain embodiments, the immunogenic compositions of the invention further comprise at least one of an adjuvant, a buffer, a cryoprotectant, a salt, a divalent cation, a non-ionic detergent, an inhibitor of free l oxidation, a t or a carrier.
The immunogenic compositions of the invention may further comprise one or more preservatives in addition to a plurality of meningococcal protein antigens and ar ccharide-protein conjugates. The FDA requires that biological products in multiple-dose (multi-dose) vials contain a vative, with only a few ions.
Vaccine products containing preservatives include vaccines containing benzethonium chloride ax), 2-phenoxyethanol (DTaP, HepA, Lyme, Polio (parenteral)), phenol (Pneumo, Typhoid (parenteral), Vaccinia) and thimerosal (DTaP, DT, Td, HepB, Hib, Influenza, JE, Mening, Pneumo, ). Preservatives approved for use in injectable drugs include, e.g., chlorobutanol, m-cresol, methylparaben, propylparaben, 2-phenoxyethanol, benzethonium chloride, benzalkonium chloride, benzoic acid, benzyl alcohol, phenol, thimerosal and phenylmercuric nitrate.
Formulations of the invention may further comprise one or more of a buffer, a salt, a divalent cation, a non-ionic detergent, a cryoprotectant such as a sugar, and an anti-oxidant such as a free radical scavenger or chelating agent, or any multiple combination f. The choice of any one component, e.g., a chelator, may determine whether or not another component (e.g., a scavenger) is desirable. The final composition formulated for administration should be sterile and/or pyrogen free. The skilled artisan may empirically determine which combinations of these and other components will be optimal for inclusion in the preservative containing immunogenic compositions of the invention depending on a variety of s such as the particular storage and stration conditions required.
In certain embodiments, a formulation of the ion which is compatible with parenteral administration comprises one or more physiologically acceptable buffers selected from, but not limited to, Tris thamine), phosphate, acetate, borate, citrate, glycine, histidine and succinate. In certain embodiments, the formulation is buffered to within a pH range of about 6.0 to about 9.0, preferably from about 6.4 to about 7.4.
In certain embodiments, it may be desirable to adjust the pH of the genic composition or formulation of the invention. The pH of a ation of the invention may be adjusted using standard techniques in the art. The pH of the ation may be ed to be between 3.0 and 8.0. In certain embodiments, the pH of the formulation may be, or may adjusted to be, between 3.0 and 6.0, 4.0 and 6.0, or 5.0 and 8.0. In other embodiments, the pH of the formulation may be, or may adjusted to be, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 5.8, about 6.0, about 6.5, about 7.0, about 7.5, or about 8.0. In certain ments, the pH may be, or may adjusted to be, in a range from 4.5 to 7.5, or from 4.5 to 6.5, from 5.0 to 5.4, from 5.4 to 5.5, from 5.5 to 5.6, from 5.6 to 5.7, from 5.7 to 5.8, from 5.8 to 5.9, from 5.9 to 6.0, from 6.0 to 6.1, from 6.1 to 6.2, from 6.2 to 6.3, from 6.3 to 6.5, from 6.5 to 7.0, from 7.0 to 7.5 or from 7.5 to 8.0. In a specific embodiment, the pH of the formulation is about 5.8.
In certain embodiments, a ation of the invention which is compatible with parenteral administration comprises one or more divalent cations, including but not limited to MgCl2, CaCl2 and MnCl2, at a concentration ranging from about 0.1 mM to about 10 mM, with up to about 5 mM being preferred.
In certain embodiments, a formulation of the invention which is compatible with eral administration comprises one or more salts, including but not limited to sodium chloride, potassium chloride, sodium sulfate, and potassium sulfate, t at an ionic strength which is logically acceptable to the subject upon eral administration and included at a final concentration to produce a selected ionic strength or osmolarity in the final formulation. The final ionic strength or osmolality of the ation will be ined by multiple components (e.g., ions from buffering compound(s) and other non-buffering salts. A preferred salt, NaCl, is present from a range of up to about 250 mM, with salt concentrations being selected to complement other components (e.g., sugars) so that the final total osmolarity of the formulation is compatible with parenteral administration (e.g., intramuscular or subcutaneous injection) and will e long term stability of the immunogenic components of the immunogenic composition formulation over various temperature ranges. Salt-free formulations will tolerate increased ranges of the one or more selected cryoprotectants to maintain desired final osmolarity levels.
In certain embodiments, a formulation of the invention which is compatible with parenteral administration comprises one or more cryoprotectants selected from but not limited to disaccharides (e.g., lactose, e, sucrose or ose) and polyhydroxy hydrocarbons (e.g., dulcitol, glycerol, mannitol and sorbitol).
In certain embodiments, the osmolarity of the formulation is in a range of from about 200 mOs/L to about 800 mOs/L, with a red range of from about 250 mOs/L to about 500 mOs/L, or about 300 mOs/L - about 400 mOs/L. A salt-free formulation may contain, for example, from about 5% to about 25% sucrose, and preferably from about 7% to about 15%, or about 10% to about 12% sucrose. Alternatively, a salt-free formulation may contain, for example, from about 3% to about 12% ol, and ably from about 4% to 7%, or about 5% to about 6% sorbitol. If salt such as sodium chloride is added, then the effective range of sucrose or sorbitol is relatively decreased. These and other such osmolality and rity considerations are well within the skill of the art.
In certain embodiments, a formulation of the invention which is compatible with parenteral administration comprises one or more free radical oxidation inhibitors and/or chelating agents. A variety of free radical scavengers and chelators are known in the art and apply to the formulations and methods of use described herein. Examples include but are not limited to ethanol, EDTA, a EDTA/ethanol combination, triethanolamine, mannitol, histidine, glycerol, sodium citrate, ol osphate, tripolyphosphate, ascorbic scorbate, succinic acid/succinate, malic acid/maleate, desferal, EDDHA and DTPA, and various combinations of two or more of the above. In certain embodiments, at least one non-reducing free radical ger may be added at a concentration that effectively es long term stability of the formulation. One or more free radical oxidation inhibitors/chelators may also be added in various ations, such as a scavenger and a divalent cation. The choice of chelator will determine whether or not the addition of a scavenger is needed.
In certain embodiments, a formulation of the invention which is compatible with parenteral administration comprises one or more nic surfactants, including but not limited to polyoxyethylene sorbitan fatty acid esters, Polysorbate-80 (TWEEN 80), Polysorbate-60 (TWEEN 60), Polysorbate-40 (TWEEN 40) and Polysorbate-20 (TWEEN 20), polyoxyethylene alkyl ethers, ing but not limited to BRIJ 58, BRIJ , as well as others such as TRITON X-100; TRITON X-114, NP40, SPAN 85 and the PLURONIC series of non-ionic surfactants (e.g., PLURONIC 121), with preferred ents Polysorbate-80 at a tration from about 0.001% to about 2% (with up to about 0.25% being preferred) or Polysorbate-40 at a concentration from about 0.001% to 1% (with up to about 0.5% being preferred).
In certain embodiments, a formulation of the invention comprises one or more additional stabilizing agents suitable for parenteral administration, e.g., a reducing agent comprising at least one thiol (-SH) group (e.g., cysteine, N-acetyl cysteine, reduced glutathione, sodium thioglycolate, thiosulfate, monothioglycerol, or mixtures thereof).
Alternatively or ally, preservative-containing immunogenic composition formulations of the invention may be further stabilized by removing oxygen from storage containers, ting the formulation from light (e.g., by using amber glass containers).
Preservative-containing immunogenic composition formulations of the invention may comprise one or more pharmaceutically able diluents, carriers or excipients, which es any excipient that does not itself induce an immune se. Suitable excipients include but are not limited to macromolecules such as proteins, saccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, sucrose (Paoletti et al, 2001, Vaccine, 19:2118), trehalose, lactose and lipid aggregates (such as oil droplets or mes). Such diluent, ent, and/or carriers are well known to the skilled artisan. Pharmaceutically acceptable excipients are discussed, e.g., in Gennaro, 2000, Remington: The Science and Practice of Pharmacy, 20th edition, ISBN:0683306472.
Compositions of the invention may be lyophilized or in s form, i.e. solutions or suspensions. Liquid formulations may advantageously be stered directly from their packaged form and are thus ideal for injection without the need for reconstitution in aqueous medium as otherwise required for lyophilized compositions of the invention.
Direct delivery of immunogenic compositions of the present invention to a subject may be accomplished by parenteral administration (intramuscularly, intraperitoneally, intradermally, subcutaneously, intravenously, or to the interstitial space of a tissue); or by rectal, oral, vaginal, topical, transdermal, intranasal, , aural, pulmonary or other mucosal administration. In a preferred embodiment, parenteral administration is by intramuscular ion, e.g., to the thigh or upper arm of the subject. Injection may be via a needle (e.g., a rmic ), but needle free injection may alternatively be used. A typical intramuscular dose is 0.5mL. Compositions of the ion may be prepared in various forms, e.g., for injection either as liquid solutions or suspensions. In certain embodiments, the composition may be prepared as a powder or spray for pulmonary administration, e.g., in an inhaler. In other embodiments, the composition may be prepared as a itory or pessary, or for nasal, aural or ocular administration, e.g., as a spray, drops, gel or powder.
Optimal amounts of ents for a particular immunogenic composition may be ascertained by standard studies involving observation of appropriate immune responses in subjects. Following an initial vaccination, subjects can receive one or several booster immunizations adequately spaced.
Packaging and Dosage Forms Immunogenic compositions of the invention may be packaged in unit dose or multi-dose form (e.g. 2 doses, 4 doses, or more). For multi-dose forms, vials are typically but not necessarily preferred over pre-filled syringes. Suitable multi-dose formats include but are not limited to: 2 to 10 doses per container at 0.1 to 2 mL per dose. In certain embodiments, the dose is a 0.5 mL dose. See, e.g., International Patent ation WO2007/127668, which is incorporated by reference herein.
Compositions may be presented in vials or other suitable storage ners, or may be presented in pre-filled delivery devices, e.g., single or multiple component syringes, which may be supplied with or without needles. A e typically but need not necessarily contains a single dose of the preservative-containing immunogenic composition of the invention, although multi-dose, pre-filled syringes are also envisioned. Likewise, a vial may include a single dose but may alternatively include multiple doses.
Effective dosage s can be routinely established, but a typical dose of the composition for injection has a volume of 0.5 mL. In certain embodiments, the dose is formulated for administration to a human subject. In certain embodiments, the dose is formulated for administration to an adult, teen, adolescent, toddler or infant (i.e., no more than one year old) human subject and may in preferred embodiments be administered by injection.
Liquid immunogenic compositions of the invention are also suitable for reconstituting other immunogenic compositions which are presented in lyophilized form.
Where an immunogenic composition is to be used for such extemporaneous reconstitution, described herein is a kit with two or more vials, two or more ready-filled syringes, or one or more of each, with the contents of the e being used to reconstitute the contents of the vial prior to ion, or vice versa.
Alternatively, immunogenic itions of the t invention may be lyophilized and reconstituted, e.g., using one of a multitude of methods for freeze drying well known in the art to form dry, regular shaped (e.g., spherical) particles, such as micropellets or microspheres, having particle characteristics such as mean diameter sizes that may be ed and controlled by g the exact methods used to prepare them. The immunogenic itions may further comprise an nt which may optionally be prepared with or contained in separate dry, regular shaped (e.g., spherical) particles such as micropellets or microspheres. In such embodiments, described herein is an immunogenic composition kit comprising a first component that includes a stabilized, dry immunogenic composition, optionally further comprising one or more preservatives described herein, and a second component comprising a sterile, aqueous solution for reconstitution of the first component. In certain embodiments, the aqueous solution comprises one or more preservatives, and may optionally se at least one adjuvant (see, e.g., WO2009/109550 porated herein by reference).
In yet r embodiment, a ner of the multi-dose format is selected from one or more of the group consisting of, but not limited to, general tory glassware, flasks, beakers, graduated cylinders, fermentors, bioreactors, tubings, pipes, bags, jars, vials, vial closures (e.g., a rubber stopper, a screw on cap), ampoules, es, dual or multi-chamber syringes, syringe stoppers, syringe plungers, rubber closures, plastic closures, glass closures, cartridges and disposable pens and the like. The ner of described herein is not limited by material of manufacture, and includes materials such as glass, metals (e.g., steel, stainless steel, aluminum, etc.) and polymers (e.g., thermoplastics, elastomers, thermoplastic-elastomers). In a particular embodiment, the container of the format is a 5 mL Schott Type 1 glass vial with a butyl stopper. The skilled artisan will appreciate that the format set forth above is by no means an exhaustive list, but merely serve as guidance to the artisan with respect to the variety of formats ble. onal formats contemplated for use in the present invention may be found in published gues from laboratory equipment vendors and manufacturers such as United States Plastic Corp. (Lima, OH), VWR.
EXAMPLES e 1: mental Procedures Serum bactericidal assay Cynomolgus es (n = 5/group) were immunized intramuscularly with rLP2086 or rP2086 (A + B) proteins adsorbed to AlPO4. Cynomolgus macaques are an example of non-human primates. Animals were vaccinated at weeks 0, 4 and 24, and ORF2086-specific IgG and functional antibody titers were determined at weeks 0, 4, 6 and 26. Serum ORF2086-specific IgG titers were determined against 6A and B.
Functional antibody titers were examined by serum bactericidal assay (SBA) against Neisseria meningitidis strains expressing either LP2086 with sequences homologous or heterologous to those contained in the e.
Serum bactericidal antibodies in macaques or rabbits immunized with ORF2086 vaccine were determined using SBAs with human complement. Rabbit immune sera or macaques immune sera were heat-inactivated to remove intrinsic complement activity and subsequently serially diluted 1:2 in Dulbecco’s PBS with Ca2+ and Mg2+ (D-PBS) in a 96-well microtiter plate to test for serum bactericidal ty against N. meningitidis s. Bacteria used in the assay were grown in GC media supplemented with Kellogg’s supplement (GCK) and monitored by optical density at 650 nm. Bacteria were ted for use in the assay at a final OD650 of 0.50-0.55, diluted in D-PBS and 1000– 3000 CFU were added to the assay mixture with 20% human complement.
Human serum with no able bactericidal activity was used as the ous complement source. Complement sources were tested for suitability against each individual test strain. A complement source was used only if the number of bacteria surviving in controls without added immune sera was >75%. Ten unique complement sources were required to perform the SBAs described in this study.
After a 30 min incubation at 37oC with 5% CO2, D-PBS was added to the reaction mixture and ts transferred to microfilter plates filled with 50% GCK media. The microfilter plates were filtered, incubated overnight at 37oC with 5% CO2 and microcolonies were stained and quantified. The serum bactericidal titers were defined as the interpolated ocal serum dilution that yielded a 50% reduction in CFU compared to the CFU in control wells without immune sera. The SBA titer is defined as the reciprocal of the interpolated dilution of test serum that causes a 50% reduction in bacterial counts after a 30min incubation at 37oC. Susceptibility to g with ORF2086 immune sera was established if there was a 4-fold or greater rise in SBA titer for 6 immune sera compared to the corresponding pre-immune sera. Sera that were negative against the assay strain at the starting dilution were assigned a titer of one half the limit of detection for the assay (i.e. 4).
Example 2: Cloning and Expression of Non-Lipidated ORF2086 ts The mature P2086 amino acid sequence corresponding to residues 27-286 from N. meningitidis strain M98250771 (A05) was originally derived from PCR amplification from genomic DNA. The forward , with a sequence of TGCCATATGAGCAGCGGAAGCGGAAG (SEQ ID NO: 22), annealed to the 5’ sequence and contained an NdeI site for cloning. The reverse primer, with a sequence of CGGATCCCTACTGTTTGCCGGCGATGC (SEQ ID NO: 23), annealed to the 3’ end of the gene and contained a termination codon TAG followed by restriction site BamHI.
The 799 bp amplified fragment was first cloned into an intermediate vector PCR2.1 (Invitrogen, Carlesbac, CA) This plasmid was d with NdeI and BamHI, and was ligated into expression vector pET9a (Novagen, Madison, WI) which had been cleaved with NdeI and BamHI. The resulting vector pLA100 (which includes SEQ ID NO: 54), expressed the mature ily A05 P2086 from strain M98250771 without the N- terminal cysteine (see SEQ ID NO: 13 wherein the N-terminal Cys at position 1 is deleted or SEQ ID NO: 55) that would be present in the lipidated protein. BLR(DE3) E. coli host strain [F- ompT hsdSB(rB-mB-) gal dcm ∆(srl-recA)306::Tn10 (TetR) (DE3)] (Novagen) was used to obtain expression of fHBP.
The same cloning steps were used to e the B02, B03, B09, B22, B24, B44, A04, A12, and A22 N-terminal Cys-deleted variants. The N-terminal Cys-containing variants were also prepared by this same method using forward s which also included the Cys codon (e.g. the first codon of SEQ ID NOs: 1-11). Based on the sequences provided herein, the skilled worker would be able to design d and reverse primers for each of these variants. For example, the following primers were used to amplify the B44 non-lipidated variant followed by cloning into pET9a using NdeI and BlpI.
Table 1 N-terminal Primer Sequence SEQ ID NO ’ TTTCTTcccgggAAGGAGatatacatatg Included—Fwd 24 TGCAGCAGCGGAGGCGGCGG 3’ ’ TTTCTTgctcagcaTTATTGC Included—Rev 25 TTGGCGGCAAGACCGAT 3’ ’ TTTCTTcccgggAAGGAGatatacatatg Deleted—Fwd 26 AGCAGCGGAGGCGGCGG 3’ ’ TTTCTTgctcagcaTTATTGC Deleted—Rev 27 TTGGCGGCAAGACCGAT 3’ Results Non-lipidated plasmid constructs were strongly expressed, but the non-lipidated protein variants were pyruvylated at the N-terminal Cys residue. See Examples 8 and 9, which describes, for example, a method for expressing the constructs. To me this lation, the inal Cys codon was deleted. See, for example, Example . Deletion of the N-terminal Cys, however, abrogated expression of the A22 and B22 variants. See e.g., Figure 4. The A05, B01, and B44 variants, however, were still expressed despite deletion of the inal Cys residue. See, for example, SEQ ID NO: 13 (A05), wherein the N-terminal Cys at position 1 is deleted, SEQ ID NO: 35 (B01 N-terminus), and SEQ ID NO: 21(B44),wherein the inal Cys at position 1 is deleted. See e.g., Figure 5. In addition, expression of the non-lipidated B09 t was not affected by deletion of the inal Cys residue. See, for example, Example 4.
Example 3: Effect of Gly/Ser Stalk on Non-Lipidated Variant Expression To determine why the A05, B01, and B44 variants were expressed in the absence of the N-terminal Cys and the A22 and B22 variants were not, the sequences of these variants were aligned. The A05, B01, and B44 variants all possess an extended series of 10 or 11 Gly and Ser residues immediately following the N-terminal Cys (i.e. Gly/Ser stalk). The A22 and B22 variants, however, only had a Gly/Ser stalk consisting of 6 Gly and Ser residues. Accordingly, the Gly/Ser stalk of the A22 and B22 variants was expanded by insertion of additional Gly and Ser residues.
Long Gly/Ser stalk variants were prepared by the methods described in Example 2 using forward primers that encode a Gly/Ser stalk with either 10 or 11 Gly and Ser The N-terminal leted, long Gly/Ser stalk (10-11 Gly/Ser residues) A22 and B22 variants showed increased expression over the inal leted A22 and B22 short Gly/Ser stalk (6 r residues) variants. These sion levels, r, were still reduced compared to the A05, B01, and B44 variant expression levels.
Example 4: Codon Optimization Expression of the non-lipidated B09 variant was not affected by deletion of the N-terminal Cys residue (see SEQ ID NO: 18, wherein the ne at position 1 is deleted, or SEQ ID NO: 49). See, e.g., Figure 6. Sequence tion of the B09 variant demonstrated that the B09 variant has a r stalk consisting of 6 Gly and Ser residues, similar to the Gly/Ser stalk of the A22 and B22 variants. Indeed, the N-terminal tails of the B09 and A22 variants are identical at the amino acid level. The N-terminal tails of the B09 and A22 variants (SEQ ID NO: 53 and 42, respectively), however, vary at the nucleic acid level by 2 nucleic acids: nucleic acids 15 and 39 of SEQ ID NO: 8. See e.g., Figure 6. The first 14 amino acids of the N-terminal tail of the B22 variant are identical to the B09 and A22 variants, and the N-terminal tail of the B22 variant only differs at the 15th amino acid. Nucleic acids 1-42 of the B22 variant are identical to nucleic acids 1-42 of the A22 variant. c acids 1-42 of the B22 variant (see SEQ ID NO: 52) are identical to c acids 1-42 of B09 (see SEQ ID NO: 53) but for differences at nucleic acids 15 and 39, when optimally aligned. Accordingly, the B22 variant differs from the B09 variant at amino acids 15 and 39 of SEQ ID NO: 8. This last sentence contains a aphical error and should state that the B22 variant differs from the B09 variant at nucleic acids 15 and 39 of SEQ ID NO: 8.
To determine if the nucleic acid ences ed the expression level of the B09 variant compared to the A22 and B22 variants, the A22 and B22 variants were mutated by point mutation to incorporate nucleic acids 15 and 39 into the corresponding codons for Gly5 and Gly13. Incorporation of these silent nucleic acid mutations significantly increased expression of the A22 and B22 N-terminal Cys-deleted variants to levels similar to the N-terminal Cys-deleted B09 variant. See e.g., Figure 7.
Accordingly, codon optimization to match the B09 variant can increase expression of N-terminal Cys-deleted pidated P2086 ts.
Further analysis of the non-lipidated variant sequences suggested additional codon optimizations in the Gly/Ser stalk to improve expression. Accordingly, additional non-lipidated variants were ucted by the method of Example 2 using forward primers comprising such codon optimized sequences. The forward primers used to generate optimized Gly/Ser stalks include any of the following ces: ATGAGCTCTGGAGGTGGAGGAAGCGGGGGCGGTGGA (SEQ ID NO: 28) M S S G G G G S G G G G (SEQ ID NO: 29) ATGAGCTCTGGAAGCGGAAGCGGGGGCGGTGGA (SEQ ID NO: 30) M S S G S G S G G G G (SEQ ID NO: 31) ATGAGCTCTGGAGGTGGAGGA (SEQ ID NO: 32) M S S G G G G (SEQ ID NO: 33) ATGAGCAGCGGGGGCGGTGGA (SEQ ID NO: 34) M S S G G G G (SEQ ID NO: 33) Example 5: Immunogenic Composition Formulation Optimization ISCOMATRIX formulated vaccines generate a rapid immune response resulting in a reduction in the number of dosages ed to achieve a greater than 4 fold response rate as measured in a serum bactericidal assay. Groups of five rhesus macaques were immunized with different formulations of a bivalent non-lipidated rP2086 vaccine. The vaccine included a non-pyruvylated non-lipidated A05 variant (SEQ ID NO: 13 wherein the N-terminal Cys at position 1 is deleted or SEQ ID NO: 55 encoded by SEQ ID NO: 54) and a non-pyruvylated non-lipidated B44 variant (SEQ ID NO: 21 wherein the N-terminal Cys at position 1 is deleted or SEQ ID NO: 44 encoded by SEQ ID NO: 51). The adjuvant units are as follows: AlPO4 is 250 mcg, ISCOMATRIX is between 10 and 100 mcg. The adjuvant units for AlPO4 shown in Tables 2-5 are shown as ram units, and are therefore shown as 0.25 (milligram) as opposed to 250 mcg.
The zation schedule was 0, 4 and 24 wks with bleeds at 0, 4, 6 and 26 weeks. There were no increases in SBA titers at post dose one for any of the . At post dose two, an se in SBA titers and the number of responders as defined by a 4 fold increase in SBA titer above baseline was ed for formulations containing the ISCOMATRIX adjuvant. Tables 2 and 3 provide the SBA GMTs observed for a fHBP Subfamily A and B strain respectively. SBA GMTs for the ISCOMATRIX formulations were 3-19 and 4 - 2 4 fold higher than those observed for the AIPO4 formulation for the A and B subfamily s respectively. Enhanced titers were also ed at post dose three for the ISCOMATRIX formulations at 13-95 and 2 - 10 for a fHBP Subfamily A and B strain respectively compared to the AIPO4 ation. Analysis of the responder rates, as defined by a four fold or greater increase in SBA titer over baseline revealed a similar trend (Tables 4 and 5).
Table 2: SBA titers (GMTs) obtained for against a MnB LP2086 Subfamily A strain immune serum from rhesus macaques immunized with different formulations of a bivalent rP2086 vaccine Adjuvant Geometric Mean titer (GMT) Vaccine lipidation AIPO4 ISCOMATRIX® wk0 wk4 wk6 wk26 0.25 - - - - + - + +++ A05/B44 - 0.25 10 - - + ++ - ++ ++++ 0.25 100 - - + +++ Five monkeys per group; Immunization schedule: 0, 4, 24 weeks; bleed schedule 0, 4, 6 and 26 wks. SBA test strain MnB M98 250771. “-“ < 8; “+” 8-32; “++” 33-128; “+++” 129-512; “++++” >512 Table 3: SBA titers (GMTs) obtained for against a MnB LP2086 ily B strain immune serum from rhesus macaques immunized with different formulations of a bivalent rP2086 vaccine nt Geometric Mean titer (GMT) Vaccine lipidation AIPO4 TRIX® wk0 wk4 wk6 wk26 0.25 - - - + +++ - +++ ++++ A05/B44 - 0.25 10 - - +++ ++++ - +++ ++++ 0.25 100 - - ++ ++++ Five monkeys per group; Immunization le: 0, 4, 24 weeks; bleed schedule 0, 4, 6 and 26 wks. SBA test strain MnB 7. “-“ < 8; “+” 8-32; “++” 33-128; “+++” 129-512; “++++” >512 Table 4: Number of rhesus macaques with a >4 fold rise in SBA Titer using a LP2086 Subfamily A strain Adjuvant No. of respondersb Vaccine lipidation AIPO4 ISCOMATRIX® wk0 wk4 wk6 wk26 0.25 - 0 0 0 2 - 10 0 0 3 5 A05/B44 - 0.25 10 0 0 2 5 - 100 0 0 4 5 0.25 100 0 0 2 5 Table 5: Number of rhesus macaques with a >4 fold rise in SBA Titer using a MnB LP2086 Subfamily B strain Adjuvant No. of respondersb Vaccine lipidation AIPO4 TRIX® wk0 wk4 wk6 wk26 0.25 - 0 0 3 5 - 10 0 0 5 5 A05/B44 - 0.25 10 0 0 5 5 - 100 0 0 4 4 0.25 100 0 0 3 5 Example 6: Immunoprotection conferred by Lipidated and Non-Lipidated Variants A inantly sed non-lipidated P2086 variant (B44) induces broad protection as measured by SBA against strains that represent diverse fHBP variants (from about 85% to about <92% ID) LP2086 sequences. These response rates were obtained for a non lipidated vaccine formulated with AlPO4. See Table 6, which shows SBA se rates to a subfamily B fHBP MnB strain generated by a bivalent fHBP vaccine. The non-lipidated vaccine (represented by a “-“ under the “lipidation” column) included 1mcg per protein of a non-pyruvylated non-lipidated A05 variant (SEQ ID NO: 13 wherein the N-terminal Cys at position 1 is deleted) and a non-pyruvylated nonlipidated B44 variant (SEQ ID NO: 21 wherein the N-terminal Cys at position 1 is deleted) .
Alternatively, a recombinantly expressed non-lipidated P2086 variant (B44) induces greater immune responses as measured by SBA titer than a ted t (B01) against strains bearing similar (>92% ID) and diverse (<92% ID) LP2086 sequences. Higher response rates (as defined by a four fold increase or greater in SBA titers over baseline) was observed for the vaccine containing the non-lipidated rP2086 B44 compared to the lipidated rLP2086 B01 vaccine (Table 6).
According to Table 6, non-lipidated B44 is a preferred subfamily B component of fHBP in a ition for providing broad coverage against (e.g., eliciting bactericidal antibodies against) le LP2086 variant strains.
Surprisingly, the inventors noted that LP2086 B09 variant strains are particularly unlikely to have positive SBA response rates with regard to heterologous (non-B09) 6 polypeptides. In particular, the inventors found that LP2086 B09 is an exception in terms of an assay strain against which the A05/B44 immunogenic composition bed in Table 6 elicited icidal antibodies. Therefore, in a preferred ment an genic composition of the invention includes a B09 ptide, in particular in the context of a composition including more than one ORF2086 subfamily B polypeptide. In a preferred embodiment an immunogenic composition that includes a non lipidated B44 may also e a non-lipidated B09 polypeptide.
Table 6: SBA response rates to a Subfamily B fHBP MnB strains generated by bivalent fHBP vaccines Immune serum from rhesus macaques.
% ID to Matched LP2086 Subfamilyfor % responders Adjuvant Variant of e lipidation pidated PD3 Wk 26 Assay Strain Vaccine Component B02 A05/B01 + 80 A05/B44 - 100 AIPO4 B03 1 + 50 0.25mg A05/B44 - 80 B09 1 + 0 A05/B44 - 0 B15 A05/B01 + 25 A05/B44 - 80 B16 A05/B01 + 0 A05/B44 - 50 B16 A05/B01 + 0 A05/B44 - 60 B24 A05/B01 + 0 A05/B44 - 60 B44 A05/B01 + 100 A05/B44 - 100 ISCOMATRIX® A05 A05/B44 - 100 100 (10 mcg) ISCOMATRIX® A05 A05/B44 - 100 100 (100 mcg) ISCOMATRIX® A22 A05/B44 - 88.9 80 (10 mcg) ISCOMATRIX® A22 A05/B44 - 88.9 100 (100 mcg) Five monkeys per group; Immunization schedule: 0, 4, 24 weeks; bleed schedule 0, 4, 6, and 26 wks.
Example 7: Codon Optimization of the B44 and B09 Variants Although the expression levels achieved in the ing examples were te for many applications, further optimization was desirable, and E. coli expression constructs ning additional codon zation over the full length of the protein were prepared and tested. One such improved sequence for expression of a non-Cys B44 protein was found to be the nucleic acid sequence set forth in SEQ ID NO: 43. As shown in Example 9, the expression construct containing SEQ ID NO: 43 showed enhanced expression compared to that of the non-optimized wild type sequence.
Expression of the N-terminal Cys deleted B09 protein was improved by applying codon changes from the above zed B44 (SEQ ID NO: 43) construct to B09 (SEQ ID NO: 48). To generate optimized B09 sequences, the B44 optimized DNA sequence (SEQ ID NO: 43) was first aligned to the DNA sequence of the B09 allele (SEQ ID NO: 48). The entire pidated coding sequence of the B09 allele (SEQ ID NO: 48) was optimized to reflect the codon changes seen in the B44 optimized allele (SEQ ID NO: 43) wherever the amino acids between B44 (SEQ ID NO: 44) and B09 (SEQ ID NO: 49) were identical. Codon sequences in the B09 allele corresponding to the identical amino acids n the B09 allele and the B44 allele were changed to reflect the codon used in the B44 optimized sequence (SEQ ID NO: 43). Codon sequences for amino acids that differ between B09 (SEQ ID NO: 49) and B44 (SEQ ID NO: 44) were not changed in the B09 DNA sequence.
Additionally, the non-lipidated B44 amino acid sequence (SEQ ID NO: 44) contains two sequential -glycine repeat ces (S-G-G-G-G)(SEQ ID NO: 56)(see also amino acids 2 to 6 of SEQ ID NO: 44) at its N-terminus, whereas the B09 allele ns only one serine-glycine repeat at the N-terminus (see amino acids 2 to 6 and amino acids 7 to 11 of SEQ ID NO: 49). The two serine-glycine repeats at the N- terminus of B44 (amino acids 2 to 6 and amino acids 7 to 11 of SEQ ID NO: 44) also have different codon usage (see nucleotides 4 to 18 and nucleotides 19 to 33 of SEQ ID NO: 43), and different combinations of the optimized B44 serine-glycine repeat (e.g., either nucleotides 4 to 18 of SEQ ID NO: 43, or tides 19 to 33 of SEQ ID NO: 43, or a combination thereof) were applied to the B09 DNA sequence (SEQ ID NO: 48, e.g., applied to nucleotides 4 to 18 of SEQ ID NO: 48) in order to examine the effect on recombinant protein expression.
Three different versions of optimized B09 were constructed: SEQ ID NO: 45 ns both serine-glycine repeats (GS1 and GS2) (nucleic acids 4 to 33 of SEQ ID NO: 43) from the optimized B44, SEQ ID NO: 46 contains GS1 (nucleic acids 4 to 18 of SEQ ID NO: 43), and SEQ ID NO: 47 contains GS2 (nucleic acids 19 to 33 of SEQ ID NO: 43). The DNA for all of the above codon optimized sequences were chemically synthesized using standard in the art chemistry. The resulting DNA was cloned into appropriate plasmid expression s and tested for expression in E. coli host cells as described in Examples 8 and 9.
Example 8: Method for sing ORF2086, B09 variant Cells of E. coli K-12 strain (derivatives of wild-type W3110 (CGSC4474) having deletions in recA, fhuA and araA) were transformed with d pEB063, which includes SEQ ID NO: 45, pEB064, which includes SEQ ID NO: 46, plasmid pEB065, which includes SEQ ID NO: 47, or plasmid pLA134, which includes SEQ ID NO: 48.
The red modifications to the K-12 strain are helpful for fermentation purposes but are not required for expression of the ns.
Cells were inoculated to a glucose-salts defined medium. After 8 hours of incubation at 37oC a linear glucose feed was applied and incubation was continued for an additional 3 hours. Isopropyl β-Dthiogalactopyranoside (IPTG) was added to the culture to a final concentration of 0.1 mM followed by 12 hours of incubation at 37C.
Cells were collected by centrifugation at 16,000xg for 10 minutes and lysed by addition of Easy-Lyse™ Cell Lysing Kit” from Lienco Technologies (St. Louis, MO) and loading buffer. The cleared lysates were analyzed for expression of B09 by Coomassie staining of SDS-PAGE gels and/or Western blot analysis with quantitation by a scanning densitometer. The results from scanning densitometry are below in Table 7: Table 7: Expression data in E. coli n Host cell Plasmid Percentage of total cell protein at 12 hours post IPTG ion, as measured by SDS-PAGE, scanning desitometry B09 E. coli K-12 pEB063 24% SEQ ID NO: 45 B09 E. coli K-12 pEB065 12% SEQ ID NO: 47 B09 E. coli K-12 pEB064 38% SEQ ID NO: 46 B09 E. coli K-12 pLA134 13% SEQ ID NO: 48 Example 9: Method for Expressing ORF2086, B44 variant Cells of E. coli B strain (BLR(DE3), Novagen) were transformed with plasmid pLN056, which includes SEQ ID NO: 51. Cells of E. coli K-12 strain ative of wildtype W3110) were transformed with plasmid pDK087, which includes SEQ ID NO: 43.
Cells were inoculated to a glucose-salts defined medium. After 8 hours of incubation at 37oC a linear glucose feed was applied and incubation was continued for an additional 3 hours. pyl β-Dthiogalactopyranoside (IPTG) was added to the culture to a final concentration of 0.1 mM followed by 12 hours of incubation at 37C. Cells were ted by centrifugation at16,000xg for 10 minutes and lysed by addition of Easy- Lyse™ Cell Lysing Kit” from Lienco Technologies (St. Louis, MO) and loading buffer.
The supermatants were analyzed for expression of B09 by COOMASSIE staining of SDS-PAGE gels and/or Western blot analysis, with quantitation by a ng densitometer. The results from scanning densitometry are below in Table 8: Table 8: Expression data in E. coli n Host cell Plasmid tage of total cell protein at 12 hours post IPTG induction, as measured by SDS-PAGE, scanning desitometry.
B44 E. coli B pLN056 1% SEQ ID NO: 51 B44 E. coli K-12 pDK087 17% SEQ ID NO: 43 Example 10: Pyruvylation The present example demonstrates that the N-terminal Cys residue of non-lipidated 6 proteins can become pyruvylated when expressed in, for example, E. coli. logous protein accumulation during production of variants A05 (SEQ ID NO: 13) and B44 (SEQ ID NO: 21) were monitored using reverse-phase high performance liquid chromatography (RP-HPLC). This separation was interfaced with a quadrupole time-of-flight mass spectrometer (QTOF-MS) to provide a means of monitoring formation of product related ts.
After being sed in the E. coli B and/or K-12 host cells, products d from these fermentations underwent a purification procedure during which a product modification was observed. Deconvolution of the mass spectra characterized the variants as exhibiting mass shifts of +70 Da, as compared to native products of 27640 and 27572 Da for A05 and B44, respectively.
Published literature indicated that a +70 Da mass shift had previously been observed in proteins and has been attributed to pyruvylation of the terminal residue.
The presence and location of the pyruvate group was med using the mass spectral fragmentation data (MS/MS). The data ted that the modification was on an amino-terminal cysteine residue, i.e., amino acid at position 1, according to A05 and B44. For A05, the percentage of pyruvylated polypeptides was about 30%, as compared to the total number of A05 polypeptides (SEQ ID NO: 13). For B44 the percentage of pyruvylated polypeptides was about 25%, as compared to the total number of B44 polypeptides (SEQ ID NO: 21).
When A05 (SEQ ID NO: 13 wherein the N-terminal Cys at position 1 is deleted or SEQ ID NO: 55) and B44 variants (SEQ ID NO: 21 wherein the N-terminal Cys at position 1 is deleted or SEQ ID NO: 44), which do not contain an amino-terminal cysteine, were ed, there was no detectable pyruvylation (+70 Da).
Example 11: Immunogenicity of B09 and B44, dually and in ation -10 groups of rhesus maccaques s were immunized with B09 variant (SEQ ID NO: 49 encoded by SEQ ID NO: 48) or B44 variant (SEQ ID NO: 44 encoded by SEQ ID NO: 43), or the A05, B09 and B44 (SEQ ID NO: 55, SEQ ID NO: 49 encoded by SEQ ID NO: 48, and SEQ ID NO: 44 encoded by SEQ ID NO: 43, respectively) formulated with 250 mcg of AlPO4 per dose. The monkeys were vaccinated via the intramuscular route at weeks 0, 4 and 8 with 10 mcg each of non-lipidated fHBP alone or in combination as listed in Table 9 and 10. Both weeks 0 and 12 serum samples were analyzed in SBAs against MnB strains with either subfamily A or subfamily B fHBP variants. Responders were recorded as animals with a 4 x rise in titer. The B44 variant tested was the optimized construct (SEQ ID NO: 43) and the broad response rates that were observed in previous studies (table above) were maintained for the optimized construct (Table 9) the B44 vaccine alone or in combination with B09. The B09 vaccine alone (Table 10) could also generate broadly cross reactive immune responses (Table 10).
Table 9: Response rates obtained for non lipidated fHBP es in rhesus macaques % ≥ 4 X Rise t Test Variant (PD3; 10 rhesus macaques per group) Vaccine (10 mcg A05 B44 B16 B24 B09 per protein; (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID (SEQ ID 13) NO: 21) NO: 60) NO: 20) NO: 18) B44 0 80 30 40 30 B44 + B09 +A05 60 80 40 50 30 Rhesus macaques (n= 10) were immunized i.m. at weeks 0, 4 and 8 with 10 mcg each of pidated fHBP alone or in combination as listed in the Vaccine column in formulation with 250 mcg of AlPO4. Both weeks 0 and 10 serum samples were analyzed in SBAs t the MnB s listed in the table. Responders are ed as animals with a 4 x rise in titer.
Table 9 indicates, for example, that a composition including a combination of non-pyruvylated non-lipidated B44, B09, and A05 showed higher cross-coverage against the test variants as compared to the coverage from a composition including B44 alone. In view of s shown in the present application, including in particular Table 6 and Table 9 together, compositions including B44, B09 and A05 alone or in combination are preferred embodiments of the present invention. In particular, compositions including both B44 and B09 are disclosed. Such composition preferably further includes a subfamily A polypeptide, such as in particular A05.
Table 10: Response rates obtained for non lipidated fHBP B09 vaccine in rhesus macaques % ≥ 4 X Rise Against Test Variant (PD3; 5 rhesus Vaccine (10 mcg per macaques per group) protein) A05 B44 B16 B24 B09 B09 40 60 40 60 60 Rhesus macaques (n= 5) were immunized i.m. at weeks 0, 4 and 8 with 10 mcg each of non-lipidated fHBP alone or in combination as listed in the Vaccine column in formulation with 250 mcg of AlPO4. Both weeks 0 and 10 serum samples were ed in SBAs against the MnB strains listed in the table. Responders are recorded as animals with a 4 x rise in titer.
Example 12: Immunoprotection conferred by Lipidated and Non-Lipidated Variants construct Twenty female New Zealand white rabbits, 2.5-3.5 kg, obtained from Charles River Canada, were pre-screened by whole cell ELISA and 10 animals were selected for this study based on their low background titers against the test strains representing fHBP variants B02 (SEQ ID NO: 16) and B44 (SEQ ID NO: 21) (Table 11). Group of three animals were i.m. immunized with 100 μg of each n formulated with 50 μg ISCOMATRIX per 0.5 ml dose at weeks 0, 4 and 9 (Table 12). Group 1 was vaccinated with non-lipidated B44 (SEQ ID NO: 44). A control group was included that was vaccinated with lipidated B01 formulated with AlP04 (250 mcg) s were bled at weeks 0, 4, 9 and 10. Individual sera from week 10 were ed and analyzed by serum bactericidal assay against multiple serogroup B ococcal strains from the fHBP B subfamily.
Table 11: Rabbits Used in The Study s: Rabbit Strain: New Zealand white Source:a Charles River Laboratory No. of Animals Per Group: 3 Total No. of Animals: 9 Age and Sex: Female Weight: 2.5-3.5 kg Table 12 rfHBP Aluminium # of (μg/0.5 Phosphate Group Variant lipidated (μg/0.5 ml animals ml 5 ml dose) dose) dose) 1 3 B44 - 100 50 2 3 B01 - 100 50 3 3 B01 + 100 - 100 Immunization schedule Weeks 0, 4, 9; Bleed schedule Weeks 0, 4, 9,10 Serum Bactericidal Assay (SBA): A microcolony -based serum bactericidal assay (SBA) against multiple serogroup B meningococcal strains (Table 13) was performed on individual serum samples. Human sera from donors were qualified as the complement source for the strain tested in the assay. Complement-mediated antibody-dependent bactericidal titers were interpolated and expressed as the reciprocal of the dilution of the test serum that killed 50% of the ococcal cells in the assay. The limit of ion of the assay was an SBA titer of 4. An SBA titer of <4 was assigned number of 2. A ≥ 4-fold rise of SBA titers in the week 10 sera in comparison to the titers in the pre-bleed was calculated and compared.
Serum bactericidal antibody activity as measured in the SBA is the logic surrogate of protection against meningococcal disease. The y of immunization with non-lipidated rfHBP to elicit bactericidal antibodies in rabbits was determined by SBA.
SBA measures the level of antibodies in a serum sample by mimicking the complement- mediated bacterial lysis that occurs lly. Rabbit serum samples collected from week 10 were analyzed by SBA against strains with a B44 fHBP or a B02 fHBP. As shown in Table 13 , one week after the third immunization (week 10), all serum samples displayed bactericidal activity against both test strains. (Table 13). The non-lipidated B44 (SEQ ID NO: 44) was more immunogenic than non-lipidated B01 in New Zealand Rabbits against these strains. The non ted B44 (SEQ ID NO: 44) formulated wit h the iscomatrix adjuvant gave comparable titers to the ted B01 formulated with aluminium phosphate t these strains. Rabbit eed sera showed generally no pre-existing bactericidal activity against the tested strains.
Table 13: Serum Bactericidal ty against fHBP Subfamily B Strains in New Zealand White Rabbits Vaccinated with Recombinant Non-lipidated fHBP GMT SBA Titer against test variant Subfamily B variant B44 (SEQ B02 (SEQ (formulation) ID NO: 21) ID NO: 16) Non lipidated B44 (SEQ ID 6675 7140 NO: 44)(ISCOMATRIX) Non lipidated B01 625 1052 (ISCOMATRIX) Lipidated B01 (AlPO4) 10099 10558 Example 13: Immunogenicity of six non-lipidated factor H g proteins in New Zealand white rabbits.
Groups of 5 rabbits were zed with non-lipidated fHBP variants as described in Table 14. Vaccines were administered at 0, 4 and 9 weeks. Rabbit serum samples collected from weeks 0 and 10 were analyzed by SBA against the strains with homologous and heterologous fHBP sequences. Table 14 shows the percent responders post the third immunization. One week after the third immunization (week ), all serum samples displayed bactericidal activity against the homologous strains as well as other test strains from the same fHBP subfamily. s pre-bleed sera showed generally no pre-existing bactericidal activity against the tested strains.
Table 14: Post Dose Three Percent of Responders in New Ze aland White Rabbits Vaccinated with Recombinant Non-lipidated fHBPs MnB Dose/0.5 0.5 mL n B09 B16 B24 B44 A05 A12 A22 fHBP mL A05 100 mcg 0.25 mg 5 100 80 100 A12 100 mcg 0.25 mg 5 100 100 100 A22 100 mcg 0.25 mg 5 80 80 80 B09 100 mcg 0.25 mg 5 100 80 60 80 B22 100 mcg 0.25 mg 5 40 100 60 100 B44 100 mcg 0.25 mg 5 0 60 40 100 A05, 100 mcg 0.25 mg 5 100 100 60 100 100 100 100 A12, each/400 B22, mcg total MnB fHBP Proteins Used A05 SEQ ID NO: 13, n the Cys at position 1 is deleted, or SEQ ID NO: 55 encoded by SEQ ID NO: 54 A12 SEQ ID NO: 14, wherein the Cys at position 1 is deleted A22 SEQ ID NO: 15, n the Cys at position 1 is deleted B09 SEQ ID NO: 18, wherein the Cys at position 1 is deleted, or SEQ ID NO: 49 encoded by SEQ ID NO: 48.
B22 SEQ ID NO: 19, wherein the Cys at position 1 is deleted B44 SEQ ID NO: 21, wherein the Cys at position 1 is d, or SEQ ID NO: 44 encoded by SEQ ID NO: 51 Test variants in Table 14: B09 B16 (SEQ B24 B44 A05 A12 A22 ID NO: 60) (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 18) NO: 20) NO: 21) NO: 13) NO: 14) NO: 15) Example 14: >non-lipidated A05 (SEQ ID NO: 55) SSGSGSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAEK TFKVGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQDHSAVVALQIE KINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPSGKAEYHGKAFSSDDAGGKLTYTIDF AAKQGHGKIEHLKTPEQNVELASAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDR AQEIAGSATVKIREKVHEIGIAGKQ >pEB042 (SEQ ID NO: 65) ATGAGCTCTGGAAGCGGAAGCGGGGGCGGTGGAGTTGCAGCAGACATTGGAACA GGATTAGCAGATGCACTGACGGCACCGTTGGATCATAAAGACAAAGGCTTGAAAT CGCTTACCTTAGAAGATTCTATTTCACAAAATGGCACCCTTACCTTGTCCGCGCAA GAAAAAACTTTTAAAGTCGGTGACAAAGATAATAGCTTAAATACAGGTAA ACTCAAAAATGATAAAATCTCGCGTTTTGATTTCGTGCAAAAAATCGAAGTAGATGG CCAAACCATTACATTAGCAAGCGGTGAATTCCAAATATATAAACAAGACCATTCAGC AGTCGTTGCATTGCAAATTGAAAAAATCAACAACCCCGACAAAATCGACAGCCTGA TAAACCAACGTTCCTTCCTTGTCAGCGGTTTGGGCGGTGAACATACAGCCTTCAAC CAATTACCAAGCGGCAAAGCGGAGTATCACGGTAAAGCATTTAGCTCAGATGATGC AGGCGGTAAATTAACTTATACAATTGACTTTGCAGCAAAACAAGGACATGGCAAAA TTGAACATTTAAAAACACCCGAACAGAACGTAGAGCTCGCATCCGCAGAACTCAAA GCAGATGAAAAATCACACGCAGTCATTTTGGGTGACACGCGCTACGGCAGCGAAG AAAAAGGTACTTACCACTTAGCTCTTTTTGGCGACCGAGCTCAAGAAATCGCAGGT AGCGCAACCGTAAAGATAAGGGAAAAGGTTCACGAAATTGGGATCGCGGGCAAAC AATAA >non-lipidated A12 (SEQ ID NO: 66) SSGGGGSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGA EKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQTITLASGEFQIYKQNHSAVVALQIEK INNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDPNGRLHYSIDFT RIEHLKTPEQNVELASAELKADEKSHAVILGDTRYGGEEKGTYHLALFGDRA QEIAGSATVKIREKVHEIGIAGKQ >pEB043(SEQ ID NO: 67) ATGAGCTCTGGAGGTGGAGGAAGCGGGGGCGGTGGAGTTGCAGCAGACATTGGA GCAGGATTAGCAGATGCACTGACGGCACCGTTGGATCATAAAGACAAAAGTTTGC AGTCGCTTACCTTAGATCAGTCTGTCAGGAAAAATGAGAAACTTAAGTTGGCGGCG CAAGGCGCTGAAAAAACTTATGGAAACGGTGACAGCTTAAATACAGGTAAACTCAA AAATGATAAAGTCTCGCGTTTTGATTTCATTCGTCAAATCGAAGTAGATGGCCAAAC 40 CATTACATTAGCAAGCGGTGAATTCCAAATATATAAACAAAACCATTCAGCAGTCGT GCAAATTGAAAAAATCAACAACCCCGACAAAATCGACAGCCTGATAAACC AACGTTCCTTCCTTGTCAGCGGTTTGGGCGGTGAACATACAGCCTTCAACCAATTA CCAGACGGCAAAGCGGAGTATCACGGTAAAGCATTTAGCTCAGATGATCCGAACG GTAGGTTACACTATTCCATTGACTTTACCAAAAAACAAGGATACGGCAGAATTGAAC 45 ATTTAAAAACGCCCGAACAGAACGTAGAGCTCGCATCCGCAGAACTCAAAGCAGAT GAAAAATCACACGCAGTCATTTTGGGTGACACGCGCTACGGCGGCGAAGAAAAAG GTACTTACCACTTAGCCCTTTTTGGCGACCGCGCTCAAGAAATCGCAGGTAGCGC AACCGTAAAGATAAGGGAAAAGGTTCACGAAATTGGGATCGCGGGCAAACAATAA ipidated A22 (SEQ ID NO: 68) SSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGN GKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKI DSLINQRSFLVSGLGGEHTAFNQLPSGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHG KIEHLKTPEQNVELASAELKADEKSHAVILGDTRYGGEEKGTYHLALFGDRAQEIAGSA TVKIREKVHEIGIAGKQ >pEB058 (SEQ ID NO: 69) ATGAGCTCTGGAGGTGGAGGAGTTGCAGCAGACATTGGAGCAGGATTAGCAGATG CACTGACGGCACCGTTGGATCATAAAGACAAAAGTTTGCAGTCGCTTACCTTAGAT CAGTCTGTCAGGAAAAATGAGAAACTTAAGTTGGCGGCGCAAGGCGCTGAAAAAA CTTATGGAAACGGTGACAGCTTAAATACAGGTAAACTCAAAAATGATAAAGTCTCG CGTTTTGATTTCATTCGTCAAATCGAAGTAGATGGCCAACTTATTACATTAGAAAGC GGTGAATTCCAAATATATAAACAAGACCATTCAGCAGTCGTTGCATTGCAAATTGAA AAAATCAACAACCCCGACAAAATCGACAGCCTGATAAACCAACGTTCCTTCCTTGT CAGCGGTTTGGGCGGTGAACATACAGCCTTCAACCAATTACCAAGCGGCAAAGCG GAGTATCACGGTAAAGCATTTAGCTCAGATGATGCAGGCGGTAAATTAACTTATAC AATTGACTTTGCAGCAAAACAAGGACATGGCAAAATTGAACATTTAAAAACACCCG AACAGAACGTAGAGCTCGCATCCGCAGAACTCAAAGCAGATGAAAAATCACACGC AGTCATTTTGGGTGACACGCGCTACGGCGGCGAAGAAAAAGGTACTTACCACTTA GCTCTTTTTGGCGACCGAGCTCAAGAAATCGCAGGTAGCGCAACCGTAAAGATAA GGGAAAAGGTTCACGAAATTGGGATCGCGGGCAAACAATAA > A62 (SEQ ID NO: 70). GenBank: ACI46789.1 CSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTY GNGDSLNTGKLKNDKVSRFDFIRQIEVDGKLITLESGEFQVYKQSHSALTALQTEQVQD SEDSGKMVAKRQFRIGDIAGEHTSFDKLPKGGSATYRGTAFGSDDAGGKLTYTIDFAA KQGHGKIEHLKTPEQNVELASAELKADEKSHAVILGDTRYGGEEKGTYHLALFGDRAQ EIAGSATVKIREKVHEIGIAGKQ >non-lipidated A62 (SEQ ID NO: 71) SSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYG TGKLKNDKVSRFDFIRQIEVDGKLITLESGEFQVYKQSHSALTALQTEQVQDS EDSGKMVAKRQFRIGDIAGEHTSFDKLPKGGSATYRGTAFGSDDAGGKLTYTIDFAAK QGHGKIEHLKTPEQNVELASAELKADEKSHAVILGDTRYGGEEKGTYHLALFGDRAQEI AGSATVKIREKVHEIGIAGKQ >pLA164 (SEQ ID NO: 72) ATGAGCAGCGGAGGGGGCGGTGTCGCCGCCGACATCGGTGCGGGGCTTGCCGA TGCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAGTCTTTAACGCTGG ATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAA AACTTATGGAAACGGCGACAGCCTTAATACGGGCAAATTGAAGAACGACAAGGTC AGCCGCTTCGACTTTATCCGTCAAATCGAAGTGGACGGGAAGCTCATTACCTTGGA GAGCGGAGAGTTCCAAGTGTACAAACAAAGCCATTCCGCCTTAACCGCCCTTCAG ACCGAGCAAGTACAAGACTCGGAGGATTCCGGGAAGATGGTTGCGAAACGCCAGT TCGGCGACATAGCGGGCGAACATACATCTTTTGACAAGCTTCCCAAAGG CGGCAGTGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCTGGCGGAAA ACTGACCTATACTATAGATTTCGCCGCCAAACAGGGACACGGCAAAATCGAACACT TGAAAACACCCGAGCAAAATGTCGAGCTTGCCTCCGCCGAACTCAAAGCAGATGA AAAATCACACGCCGTCATTTTGGGCGACACGCGCTACGGCGGCGAAGAAAAAGGC ACTTACCACCTCGCCCTTTTCGGCGACCGCGCCCAAGAAATCGCCGGCTCGGCAA CCGTGAAGATAAGGGAAAAGGTTCACGAAATCGGCATCGCCGGCAAACAGTAA > pDK086 (SEQ ID NO: 73) ATGTCCAGCGGTTCAGGCAGCGGCGGTGGAGGCGTGGCAGCAGATATCGGAACA GGTTTAGCAGATGCTCTGACAGCACCCTTAGATCACAAAGACAAAGGACTTAAATC ACTGACATTGGAAGATTCTATCTCGCAAAATGGTACTCTCACTCTTTCAGCCCAAG GCGCAGAAAAAACATTTAAAGTAGGCGATAAAGATAACTCCTTAAATACAGGTAAAT TAAAAAATGACAAAATCTCACGGTTTGATTTCGTTCAGAAAATTGAAGTAGATGGAC AAACGATTACATTAGCAAGCGGCGAATTCCAAATTTATAAACAAGACCATTCAGCA GTAGTAGCATTACAAATCGAAAAAATTAACAACCCGGACAAAATTGATTCTCTTATT AACCAACGCTCTTTTCTCGTATCAGGACTTGGTGGTGAACATACAGCGTTTAATCA ACTGCCGTCAGGAAAAGCAGAATATCATGGTAAAGCATTTTCATCAGACGACGCAG GTGGCAAACTGACCTATACTATTGACTTTGCAGCAAAACAGGGACATGGAAAAATT GAACATTTAAAAACACCCGAACAGAACGTAGAACTGGCCTCAGCAGAATTGAAAGC TGATGAAAAATCCCATGCAGTAATTTTAGGCGATACACGTTACGGTAGCGAAGAAA AAGGTACATATCACTTAGCTCTTTTTGGCGATCGTGCTCAAGAAATTGCTGGTTCC GTTAAAATCCGTGAAAAAGTACATGAAATCGGCATTGCAGGTAAACAATA >A29 (SEQ ID NO: 74) CSSGGGGSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDSIPQNGTLTLSAQGA EKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQNHSAVVAL QIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPGDKAEYHGKAFSSDDPNGRLHYT QGYGRIEHLKTPELNVDLASAELKADEKSHAVILGDTRYGSEEKGTYHLALFG 40 DRAQEIAGSATVKIGEKVHEIGIAGKQ >non-lipidated B22 (SEQ ID NO: 75) SSGGGGVAADIGAVLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGN GDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQTEQVQDSE HSGKMVAKRQFRIGDIAGEHTSFDKLPEGGRATYRGTAFGSDDASGKLTYTIDFAAKQ GHGKIEHLKSPELNVDLAASDIKPDKKRHAVISGSVLYNQAEKGSYSLGIFGGQAQEVA GSAEVETANGIRHIGLAAKQ >non-lipidated A05 (SEQ ID NO: 76) (pPW102) CGSSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAEKTF KVGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQDHSAVVALQIEKI NNPDKIDSLINQRSFLVSGLGGEHTAFNQLPSGKAEYHGKAFSSDDAGGKLTYTIDFAA KQGHGKIEHLKTPEQNVELASAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQ EIAGSATVKIREKVHEIGIAGKQ >non-lipidated A05 (SEQ ID NO: 77) GSSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAEKTFK VGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQDHSAVVALQIEKIN NPDKIDSLINQRSFLVSGLGGEHTAFNQLPSGKAEYHGKAFSSDDAGGKLTYTIDFAAK QGHGKIEHLKTPEQNVELASAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEI KIREKVHEIGIAGKQ >Consensus (SEQ ID NO: 78) CSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTY GNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQSHSALVALQTEQINNS DKSGSLINQRSFRISGIAGEHTAFNQLPKGGKATYRGTAFSSDDAGGKLTYTIDFAAKQ HLKTPEQNVELASAELKADEKSHAVILGDTRYGGEEKGTYHLALFGDRAQEIA GSATVKIREKVHEIGIAGKQ >Consensus (SEQ ID NO: 79) VAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYG NGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQSHSALVALQTEQINNSD KSGSLINQRSFRISGIAGEHTAFNQLPKGGKATYRGTAFSSDDAGGKLTYTIDFAAKQG HGKIEHLKTPEQNVELASAELKADEKSHAVILGDTRYGGEEKGTYHLALFGDRAQEIAG SATVKIREKVHEIGIAGKQ Example 15: Generation of non-lipidated variants of subfamily A rP2086- Cloning of non lipidated fHBP genes The coding sequence of non ted A05 fHBP protein (SEQ ID NO: 55) was aligned to an expression-optimized B44 ce (SEQ ID NO: 43). Wherever the amino acids between the two were identical, the codon from the B44 (SEQ ID NO: 43) was used to substitute in the A05 gene. The optimized sequence was synthesized de novo at Celtek Genes, adding restriction endonuclease sites NdeI and BamHI at the N- and C-termini, respectively. The resulting gene (SEQ ID NO: 65) was subcloned into pET30a at those sites.
Recombinant non lipidated A12 fHBP (SEQ ID NO: 66) was expressed from pEB043 (SEQ ID NO: 67). The A12 allele was expression -optimized by Blue Heron Technologies. This gene was optimized by the same process as for A05 (pEB042). In addition, the Blue Heron optimized B44 SGGGGSGGGG (amino acid residues 2 to 11 of SEQ ID NO: 44) amino al codons replaced the native A12 SSGGGG (amino acid es 1 to 6 of SEQ ID NO: 66) codons. The optimized sequence was sized de novo at Celtek Genes, adding restriction endonuclease sites NdeI and BamHI at the N- and C-termini, respectively. The resulting gene (SEQ ID NO: 67) was subcloned into pET30a at those sites.
Recombinant non lipidated A22 fHBP (SEQ ID NO: 68) was expressed from pEB058 (SEQ ID NO: 69). This gene was optimized by the same process as for pEB042. In addition, the Blue Heron optimized B44 SGGGG (amino acid residues 2 to 6 of SEQ ID NO: 44) amino al codons ed the native A22 SSGGGG (amino acid residues 1 to 6 of SEQ ID NO: 68) codons. The optimized sequence was synthesized de novo at Celtek Genes, adding ction endonuclease sites NdeI and BamHI at the N- and C-termini, respectively. The resulting gene (SEQ ID NO: 69) was subcloned into pET30a at those sites.
Recombinant A62 fHBP (SEQ ID NO: 71) was expressed from pLA164 (SEQ ID NO: 72). The A62_002 allele from strain 0167/03 was PCR amplified with primers containing restriction endonuclease sites NdeI and BamHI at the N- and C-termini, respectively. The resulting gene (SEQ ID NO: 72) was subcloned into pET30a at those sites.
Example 16: Expression, Fermentation, and Purification of Subfamily A rP2086 ns E. coli expression strains BLR(DE3) E. coli B recA- transformed with pLA164 (SEQ ID NO: 72) was used for expression of A62 (SEQ ID NO: 71). Plasmid pEB042 (SEQ ID NO: 65) was transformed to E. coli host BD643 (W3110:DE3 ΔrecA ΔfhuA ΔaraA) to give strain BD660 for expression of A05 (SEQ ID NO: 55). Expression of A22 (SEQ ID NO: 68) was from strain BD592 which consists of plasmid pEB058 (SEQ ID NO: 69) residing in host BD559 (which is also W3110:DE3 ΔrecA ΔfhuA . Lastly, plasmid pEB043 (SEQ ID NO: 67) was transformed to host BD483 :DE3 ΔrecA) to give strain BD540 for expression of A12 (SEQ ID NO: 66).
Fermentation sion strains were fermented in a glucose-based minimal . An overnight starter culture was inoculated to ten liter fermentors operated at 37oC, 1vvm aeration with cascade dO control at 20%. When batched glucose was exhausted from the medium (at ~OD600=15) a limiting linear glucose feed at 3.8 g/L/hr was initiated. The feed was continued up to induction with 0.1mM IPTG and through the subsequent protein expression period. For expression of A05 (SEQ ID NO: 55), strain BD660 was induced at OD600=25 and fermentation was continued through 7 hours nduction (HPI). Expression of A22 (SEQ ID NO: 68) and A12 (SEQ ID NO: 66)from strains BD592 and BD540, respectively, was achieved by inducing at OD600=40 and fermenting for 24 HPI. At the end of the fermentation, cell pastes were collected by centrifugation.
A62 (SEQ ID NO: 71) rP2086 proteins are produced as soluble proteins in the cytoplasm of E.coli strains. The soluble cytoplasmic extract is typically obtained by thawing frozen cells sing a particular variant of the subfamily A of rP2086 in hypotonic buffer (10mM Hepes-NaOH pH 7.4 containing protease inhibitors) and disrupting the cells in a Microfluidizer under ~20,000 psi. RNase and DNAse are added to digest nucleic acids and the cytoplasmic t is collected as the supernatant following centrifugation at low speed to remove any en cells and then high speed (>100,000xg) to remove membranes, cell walls and other larger lular components. The cytoplasmic extract is further clarified by sequential ments to 25% then 50% saturated ammonium e and removal of precipitated material after each adjustment by low speed centrifugation. Low lar weight ionic cell ents are then removed by adsorbing the rP2086 in 50% ammonium saturated sulfate in a buffer of 10mM Hepes- NaOH pH7.4, 1mM Na2EDTA to a hydrophobic interaction column (phenyl sepharose purchased from GE Healthcare) then eluting the rP2086 by linearly sing the ammonium sulfate concentration to 0% with a buffer of 10mM Hepes-NaOH pH7.4, 1mM A. The majority of the vely d proteins are then removed by adjusting the rP2086 containing fractions to a buffer of 10mM Tris-HCl, pH 8.6, 1mM Na2EDTA passage of the pooled fractions over an anion exchange column (TMAE purchased from EMD) brated with the same buffer. The rP2086 is then further purified by chromatography on ceramic hydroxyapatite (obtained from ) by exchanging the buffer containing the rP2086 to 10mM Hepes-NaOH, pH7.4 containing 1mM sodium phosphate adsorbing the protein to the ceramic hydroxyapatite then eluting with a linear gradient of sodium phosphate to 250mM at pH 7.4. The unit operations listed above are often sufficient to yield purified rP2086 ily A members. However, since the expression level can vary over 10-fold, when the rP2086 is expressed at the lower end of the range or when ultra pure rP2086 is need (at high concentrations for NMR structural determinations) the following additional unit operations are added: chromatofocusing followed by ceramic hydroxyapatite chromatography. The buffer containing rP2086 protein from the earlier hydroxyapatite step is exchanged to 25mM Tris-acetate, pH8.3 and the protein is adsorbed to a chromatofocusing PBE94 column (obtained from GE Healthcare) equilibrated with the same buffer and then eluted with a buffer of polybuffer 94-acetate, pH 6. The rP2086 proteins will elute at their ~pI and the fractions containing the protein are pooled. The buffer of the rP2086 containing fractions is then exchanged to 10mM Hepes-NaOH pH7.4 containing 1mM sodium phosphate and adsorbed and eluted as above. The rP2086 ily A members prepared by this s are typically >95% homogeneous by SDS-PAGE analysis and most often >99% homogeneous.
A05, A12 and A22 (SEQ ID NOs: 55, 66, and 68, respectively) At the end of fermentation, the cell slurry is recovered by continuous centrifugation and re-suspended to ~1/4 the original fermentation volume in 20 mM Tris, mM EDTA, pH 6.0. Lysis of the cell suspension is achieved by ressure nization (2 passes, 4000-9000 psi). To the nate is added DADMAC to a final concentration of 0.5%. The solution is stirred at 15-25 ºC for 60 minutes during which time a heavy precipitate forms. The solution is clarified by continuous centrifugation. The proteins (A05, A12 and A22) are ed using two chromatographic steps followed by a final buffer exchange. The pH of the centrate is adjusted to 5.5 and loaded onto a GigaCap-S column (CEX). The protein binds to the resin and is subsequently eluted using a sodium chloride gradient. To the pool from the CEX column is added sodium citrate to a final concentration of 1.5 M, and the solution is loaded onto a Phenyl-650M column (HIC). The protein binds to the resin and is subsequently eluted using a sodium citrate step gradient. The HIC pool ning purified protein is exchanged into the final drug nce buffer by diafiltration. A 5 kD regenerated cellulose acetate ultrafiltration cassette is utilized. The protein concentration is targeted to 1.5-2.0 mg/mL. The diafiltered retentate is filtered through a 0.2 micron filter prior to filling into the storage bottles. Drug substance is stored at -70ºC.
Example 17: Serum bactericidal assay Functional antibody titers were examined by serum bactericidal assay (SBA) t wildtype or engineered Neisseria meningitidis serogroup B strains expressing fHBP either with sequences homologous or heterologous to those contained in the vaccine. Serum bactericidal antibodies in rabbits immunized with rP2086 vaccines were determined using SBAs with human complement. Rabbit immune sera was heat-inactivated to remove intrinsic complement activity and subsequently serially diluted two-fold in Dulbecco’s PBS with Ca2+ and Mg2+ (D-PBS) in a 96-well iter plate to test for serum bactericidal activity against N. itidis s. For combination studies with engineered strains, sera of interest was mixed in a 1:1 ratio before the serial dilution described above, so the effective concentration of each component was half that when each was tested dually. Bacteria used in the assay were grown in GC media mented with Kellogg’s ment (GCK) and red by optical density at 650 nm. Bacteria were harvested for use in the assay at a final OD650 of 0.50-0.55, diluted in D-PBS and 1000–3000 CFU were added to the assay mixture. Human serum with no detectable bactericidal activity was used as the exogenous ment source. Complement sources were tested for suitability against each individual test strain. For the isogenic strains, a single human serum was identified and qualified for SBAs against all isogenic strains. A complement source was used only if the number of bacteria surviving in controls without added immune sera was >75%. After a 30 minute incubation at 37oC with 5% CO2 and an agitation of 700 rpm on a shaker, D-PBS was added to the reaction mixture and aliquots transferred to microfilter plates prefilled with 50% GCK media for the wild type strains and 100% GCK media for the engineered strains. The microfilter plates were filtered, incubated overnight at 37oC with 5% CO2 and microcolonies were stained and quantified. The serum bactericidal titers were defined as the olated reciprocal serum dilution that yielded a 50% reduction in CFU compared to the CFU in control wells t immune sera. Susceptibility to killing by anti-rP2086 immune sera was established if there was a 4-fold or greater rise in SBA titer for anti-rP2086 immune sera ed to the corresponding pre-immune sera. Sera that were negative against the assay strain at the starting dilution were assigned a titer of one half the limit of detection for the assay.
Example 18: genicity of non-lipidated variants of rP2086 sub family A proteins White New Zealand female rabbits (2.5-3.5 kg) obtained from Charles River (Canada) were used in two studies. For the first study, groups of 3 rabbits were immunized with either 30 mcg or 3 mcg each of either a lipidated A05 or a non-lipidated A05 fHBP formulation. For the second study, five rabbits/group were immunized intramuscularly at the right hind leg with with rP2086A variants at 20 µg/mL adjuvanted with 500 µg/mL of AlPO4 (0.5ml/dose/two sites). Animals were vaccinated at weeks 0, 4 and 9, bled at weeks 0 and 6 and exsanguinated at week 10. LP2086 specific bactericidal antibody titers were determined at weeks 0, 6 and 10.
The goal of these studies was to mimic the d responses that are observed for immunologically naïve populations such as infants. First we compared a low and high dosage (30 vs 3 mcg per antigen per dose) of vaccines containing either lipidated A05 (SEQ ID NO: 13) or non-lipidated A05 (SEQ ID NO: 55) s 15 A and 15B).
Low dosages were used so that differences in the response rate could be discerned n each vaccine. SBA analysis was conducted using two strain sets. The first set ted of wildtype strains that had caused invasive disease. The second was a genetically engineered strain set that had the same strain background and differed only by the sequence of the fHBP being expressed as follows: the N. menigitidis strain PMB3556, which expresses a B24 variant of fHBP, was engineered such that its endogenous fhbp gene was replaced with genes encoding for other fHBP variants. The constructs were designed such that only the region encoding the ORF was “switched” and the surrounding genetic background was left intact. SBA analysis using this strain set therefore allowed for evaluation of reactivity against different subfamily A fHBP ns expressed at the same level and in the same genetic background using one source of human complement. All strains had fHBP expression levels that were above the threshold identified by Jiang et al (2010). As shown in Tables 15A and 15B, both the high and low dose levels of the lipidated A05-containing vaccine elicited broad protection across the genetically diverse subfamily A variants, s reduced responses were observed at both doses for the vaccine containing the non-lipidated A05 variant. This side-by-side comparison therefore revealed that, although the nonlipidated A05 variant is cross tive across ily A expressing s, it is not as immunogenic as the lipidated variant which is more likely to form a native configuration (Tables 15A and 15B).
For the subsequent study, the dose level was raised to 10 mcg per non-lipidated ily A variant to assess each for its potential to provide broad coverage against subfamily A strains. SBA analysis reveal that at this raised dose level sera from rabbits immunized with non-lipidated A05 (SEQ ID NO: 55), A62 (SEQ ID NO: 71), A12 (SEQ ID NO: 66) and A22 (SEQ ID NO: 68) fHBP variants all induced titers to wildtype strains expressing both homologous and heterologous subfamily A variants, indicating that all were cross-protective at this low dose within subfamily A. Therefore we observed that the N2C1 vaccine (A05) could generate antibodies that could kill the N1C2 (A22) and N2C2 (A12) variant strains and likewise vaccines from these other groups could kill strains with opposing variants. Under these conditions, it was observed that the A05 and A62 variants induced the highest SBA responder rates across strains (Table 16). ingly, this shows a protective effect across these ts.
Table 15A- Lipidated A05 ation Geometric Mean SBA Titers Lipidated A05 formulation mcg dose 3 mcg dose fHBP variant strain name pre PD3 4xrise pre PD3 4xrise Wildtype A05 PMB1745 2 697 3 2 382 3 A12 PMB258 5 406 3 2 99 3 A22 PMB3570 2 956 3 3 185 3 A62 PMB3037 2 959 3 2 50 3 Isogenic A05 RD3040-A05 102 3424 3 38 583 3 strains A12 RD3044-A12 15 1233 3 8 183 3 A22 RD3042-A22 24 3289 3 6 582 3 A29 RD3043-A29 63 4086 3 19 1359 3 Table 15B- Non-lipidated A05 formulation Geometric Mean SBA Titers pidated A05 formulation mcg dose 3 mcg dose fHBP strain name pre PD3 4xrise pre PD3 4xrise Wildtype A05 PMB1745 2 1182 3 2 281 3 strains A12 PMB258 5 31 2 6 23 1 A22 PMB3570 2 76 3 2 11 2 A62 PMB3037 2 35 2 2 2 0 Isogenic A05 RD3040-A05 95 258 0 78 134 1 strains A12 RD3044-A12 34 228 2 50 105 1 A22 -A22 24 221 2 23 85 1 A29 RD3043-A29 36 326 3 52 267 2 Tables 15A and 15B. Geometric Mean SBA Titers against N. meningitidis group B s of sera taken pre and post (PD3 = 10 weeks) immunization of rabbits (n = 3) with either 30 or 3 mcg vaccines containing lipidated or non-lipidated A05. The upper panels ed “wildtype strains”) of Tables 15A and 15B summarizes activity against clinical isolates. The lower panels (labeled “isogenic strains”) of Tables 15 A and 15B summarizes activity against a set of isogenic strains which were engineered from the parental N. meningitidis strain ( PMB3556) such that the entire ORF of its endogenous fHBP was replaced with either A05 (SEQ ID NO: 13), A22 (SEQ ID NO: 15), A29 (SEQ ID NO: 74) or A12 (SEQ ID NO: 14) variants.
Percent of Responders with >4 fold rise vaccine A05 A62 A12 A22 e A62 100 100 60 60 80 A05 80 80 60 80 75 A12 60 80 60 60 65 A22 60 60 40 40 50 Table 16. The percentage of responders trating at least 4-fold rise in SBA GMT levels over background from 10 week sera taken from rabbits immunized with 10 mcg of non-lipidated A subfamily fHBP variants against strains expressing A05, A62, A12 or A22 fHBP variants.
Cross-protection was also observed for all variants using the ic strain set described above at the increased dose of 10 mcg, with sera from rabbits immunized with the A62 variant (SEQ ID NO: 71) demonstrating the most cross-reactivity, followed by A05 anti-sera (Table 17). In addition, sera from rabbits immunized with the A62 variant (SEQ ID NO: 71) showed reactivity to both the al PMB3556 strain and the B09 switched strain (Table 18), indicating that cross-reactivity activity extends to subfamily B proteins. A62 appears to be ed of both subfamily A (A22) and subfamily B (B09) domains (Figure 9).
Geometric Mean SBA Titers vs. Isogenic Strain Set RD3040- RD3042- - RD3044- PMB3556 (B24 KA3011 A05 A22 A29 A12 parent) Vaccine pre PD3 pre PD3 pre PD3 pre PD3 pre PD3 pre PD3 A62 17 36 31 69 4 95 23 45 44 109 4 2 A05 7 67 5 64 20 132 16 58 34 40 3 2 A12 12 40 8 34 3 40 25 149 27 46 3 2 A22 9 46 13 36 5 30 13 38 28 34 4 2 Percent of Responders (4-fold rise) Vaccine RD3040-A05 RD3042-A22 RD3043-A29 RD3044-A12 PMB3556 KA3011 A62 40 80 100 40 40 0 A05 80 80 60 40 0 0 A12 40 40 60 60 20 0 A22 80 40 60 60 20 0 Table 17. Isogenic “switched” strains were engineered from the parental N. meningitidis strain 56) such that the entire ORF of its endogenous fHBP (a B24 variant) was ed with either A05 (SEQ ID NO: 13), A22(SEQ ID NO: 15), A29 (SEQ ID NO: 74) or A12 (SEQ ID NO: 14) ts. KA3011 is a negative control strain (i.e. the parental PMB3556 whose fhbp gene has been deleted). The Geometric Mean SBA Titers (n = ) of sera (taken before or 10 weeks after immunization of rabbits with three doses of mcg non-lipidated A subfamily fHBP variants) against these strains is shown in the upper panel. The percentage of responders demonstrating at least a 4-fold rise in response over background is shown in the lower panel.
Geometric mean SBA titers t isogenic subfamily B strains PMB3556 (parent) RD30337-B09 Vaccine pre PD3 %responders pre PD3 %responders ld rise) (>4-fold rise) A62 44 109 60 31 163 60 A05 34 40 0 32 28 0 A12 27 46 20 19 23 20 A22 28 34 0 29 30 0 Table 18. The Geometric Mean SBA Titers of sera (taken before or 10 weeks after immunization of rabbits (n = 5) with 10 mcg non-lipidated subfamily A proteins (A62 (SEQ ID NO: 71); A05 (SEQ ID NO: 55); A12 (SEQ ID NO: 66); A22 (SEQ ID NO: 68)) against two subfamily B isogenic strains.
Example 19: Evaluation of the effect of combining sera raised against nonlipidated subfamily A proteins on SBA Combinations of serum were assessed to evaluate the effect on the breath of coverage. Paired pre vs post ation serum were tested to confirm that there was no non-specific killing d as a result of combining the serum. The GM fold rise was calculated for the individual sera and for the combinations of serum across the 4 isogenic strains that represented diversity within subfamily A. Fold rise increases were detected for some of the combinations tested providing evidence that the breadth of ge can be increased by including more subfamily A variants (Table 19). Optimal combinations appear to be A05 (SEQ ID NO: 55) with A62 (SEQ ID NO: 71) or A62 (SEQ ID NO: 71) with A12 (SEQ ID NO: 66) (Table 20).
BC50 titer A05 A12 A62 AQ508-5 AQ509-4 AQ507-5 Strain Wk0 Wk10 Fold Wk0 Wk10 Fold Wk0 Wk10 Fold rise rise rise RD3040- 2 98 49 2 65 33 3 14 5 RD3042- 2 116 58 2 94 47 2 81 40 - 3 368 123 2 198 99 5 54 11 RD3044- 2 37 19 3 486 162 3 45 15 GM fold rise 50 70 13 KA3011 2 2 1 2 2 1 9 5 1 BC50 titer A05 + A12 A05 + A62 A12 + A62 + AQ509-4 AQ508-5 + AQ507-5 AQ509-4 + 5 Strain Wk0 Wk10 Fold Wk0 Wk10 Fold Wk0 Wk10 Fold rise rise rise RD3040- 7 170 24 8 107 13 2 97 49 RD3042- 6 3418 570 6 160 27 2 181 91 RD3043- 2 509 255 7 1181 169 6 478 80 RD3044- 8 335 42 5 1302 260 7 3707 530 GM fold rise 110 63 117 KA3011 13 2 0 2 5 3 7 5 1 Table 19. SBA Titers of sera from the highest responders of each vaccine group were retested against the isogenic strain set as shown in Table 17. Sera was tested in one to one mixtures to determine the extent of synergistic activity.
Fold Rise Increase for Combination Vaccine vs Combination Monovalent A05 A12 A62 A05 (SEQ ID NO: 55) + Q ID NO: 66) 2.2 1.6 A05 (SEQ ID NO: 55) + A62 (SEQ ID NO: 71) 1.3 4.8 A12 (SEQ ID NO: 66)+ A62 (SEQ ID NO: 71) 1.7 8.9 Table 20. The fold rise se for sera tested in combination as compared to each tested alone (calculated from Table 19).
The results presented above in Examples 18-19 show that non-lipidated subfamily A proteins are immunogenic and may provide protection against infection with N. itidis strains bearing either homologous or heterologous variants. The data presented here illustrates that selected non-lipidated subfamily A variants retain immunogenicity and provide protection against heterologous strains, though these responses are lower than the lipidated variants. We also demonstrate that the A62 (SEQ ID NO: 71) rP2086 antigen, having sequence similarity to subfamily B (see, for example, Figure 9), may protect across the subfamilies because the A62 (SEQ ID NO: 71) vaccine may kill strains expressing ily B variants B09 or B24).
The data presented above shows that not only are non-lipidated subfamily A variants capable of the type of y observed with combinations of lipidated fHBP, but also that they may e coverage against B subfamily variants.
Example 20: Evaluation of immunogenicity of the combination of factor H binding proteins and tetravalent meningococcal conjugate vaccine in New Zealand white s The study was carried out in New Zealand White rabbits in the 2.5-3.5 kg range obtained from Charles River, Canada (Table 21). Prior to entering the study, 55 rabbits were pre-screened for existing antibodies using whole cell ELISAs t strains A05 and B02. After the screening, the s with relatively low antibody titers (specific IgG titers <350) were vaccinated intramuscularly at the hind legs, 0.5 mL per site (1.0mL per dose, see Table 22) at weeks 0, 4, and 9. There were three rabbits per group. Rabbits were bled at weeks 0, 4, 6, 9, and exsanguinated at week 10. Serum samples were prepared and week 0 and 10 serum s were analyzed by SBA. The meningococcal conjugate vaccine O®, meningococcal (Groups A, C, Y and W- 135) oligosaccharide diphtheria CRM197 conjugate vaccine, is), bivalent rLP2086 and tetravalent non-lipidated variants and their combinations were prepared according to 23-26.
Table 21:Rabbits Used in This Study Species: Rabbit Strain: New Zealand white Source:a Charles River Laboratory No. of Animals Per Group: 3 Total No. of Animals: 30 Age and Sex: Male Weight: 2.5-3.5 kg a Rabbits were maintained in accordance with the established Institutional Animal Care and Use Committee guidelines.
The design of the study is shown in Table 22.
Table 22: Experimental Design Group # of Immunogen nt Vax Serum Rabb (wk) Prep 1 3 1 Human Dosage None 0, 4, Wk 0, 4, 6, MENVEO/dose 1.0 mL/2 9 9 sites Exsang: Wk 10 2 3 1:10 Human Dosage None 0, 4, Wk 0, 4, 6, MENVEO/dose 1.0 mL/2 9 9 sites Exsang: Wk 10 3 3 1 Human Dosage MENVEO AlPO4 0, 4, Wk 0, 4, 6, + 30 µg rLP2086-A (A05 250 9 9 (SEQ ID NO: 13)) + 30 µg µg/dose/1.0mL Exsang: rLP2086-B (B01 (SEQ ID Wk 10 NO: 58))/dose 1.0 mL/2 sites 4 3 1:10 Human Dosage AlPO4 0, Wk 0, 4, 6, MENVEO + 3 µg 6-A 250 4, 9 9 (A05 (SEQ ID NO: 13)) + µg/dose/1.0mL Exsang: 3 µg rLP2086-B (B01 (SEQ Wk 10 ID NO: 58))/dose 1.0 mL/2 sites 3 30 µg rLP2086-A (A05 AlPO4 0, 4, Wk 0, 4, 6, (SEQ ID NO: 13))+ 250 9 9 µg rLP2086-B (B01 µg/dose/1.0mL Exsang: (SEQ ID NO: 58)/dose 1.0 Wk 10 mL/2 sites 6 3 3 µg rLP2086-A (A05 (SEQ AlPO4 0, 4, Wk 0, 4, 6, ID NO: 13))+ 3 µg rLP2086- 250 9 9 B (B01 (SEQ ID NO: µg/dose/1.0mL Exsang: 58)/dose 1.0 mL/2 sites Wk 10 7 3 Non-Lipidated -A05 AlPO4 0, 4, Wk 0, 4, 6, (SEQ ID NO: 55), B09 (SEQ 250 9 9 ID NO: 49), B22 (SEQ ID µg/dose/1.0mL : NO: 75), and B44 (SEQ ID Wk 10 NO: 44), 30 µg each/dose 1.0 mL/2 sites 8 3 Non-Lipidated rP2086-A05, AlPO4 0, 4, Wk 0, 4, 6, B09, B22, and B44, 3 µg 250 9 9 each/dose 1.0 mL/2 sites µg/dose/1.0mL Exsang: Wk 10 9 3 1 Human Dosage MENVEO AlPO4 0, 4, Wk 0, 4, 6, + Non-Lipidated rP2086- 250 9 9 A05, B09, B22, and B44, 30 µg/dose/1.0mL Exsang: µg each/dose 1.0 mL/2 sites Wk 10 3 1:10 Human Dosage of AlPO4 0, 4, Wk 0, 4, 6, MENVEO + Non-Lipidated 250 9 9 rP2086-A05, B09, B22, and µg/dose/1.0mL Exsang: B44, 3 µg each/dose 1.0 Wk 10 mL/2 sites Summary of ations Table 23: Formulations for Immunization Amount Presentation/ Material Function ation Provided for Appearance 3 doses MENVEO® ococcal (Groups A, C, Y Lyo A: White, and W-135) Novartis product fluffy cake oligosaccharide contains Liquid C, Y, W- eria Active Meningococccal 3 x 15 doses 135: Clear, CRM197 groups A, C, Y and colorless conjugate W-135 solution vaccine, Novartis rLP2086 subfamily A rLP2086-A and B at 120 µg/mL White to off (A05 (SEQ ID 3 x 15 per protein in white NO: 13)), syringes Active Histidine pH 6.0, homogeneous rLP2086-B (0.57mL fill appox 0.005% PS80 cloudy (B01 (SEQ ID volume) with 0.5 mg/mL Al of suspension NO: 58)) AlPO4 A05 (SEQ ID NO: 55), B44 (SEQ ID NO: 44), B22 (SEQ ID NO: 75), and B09 L44857-50 (SEQ ID NO: 49) at 3 x 15 vials Lyophilized; MnB tetravalent Active 0.6 mg/mL (0.7mL recon white fluffy cake non-lipidated formulated in 10 mM volume) Histidine buffer, pH 6.5 with 0.01% PS80, 4.5% Trehalose, and mL 0.5 White to off mg/mL in 3 white AlPO4, 60 mM NaCl, glass vials AlPO4 Adjuvant homogeneous WFI 30 mL 0.25 cloudy mg/mL in 3 suspension glass vials 3 x 20 vials Clear, colorless 60 mM Saline Diluent NA (1.0 mL fill volume) Table 24: ents and Container/Closure Information Formulation Lot # Source Excipients MENVEO® MenCYW-135 Liquid Novartis The vaccine contains no Conjugate Component preservative or adjuvant. (091101) Each dose of vaccine MenA Lyophilized ns 10 μg MenA Conjugate Component oligosaccharide, 5 μg of (029011) each of MenC, MenY and MenW135 oligosaccharides and 32.7 to 64.1 μg CRM197 protein. Residual formaldehyde per dose is estimated to be not more than 0.30 μg.
(Unknown previously). rLP2086-A D007 v1.0 CSMD, Pfizer Histidine pH 6.0, appox (A05 (SEQ ID Pearl River, 0.005% PS80, 0.5 mg/mL NO: 13)), NY Al of AlPO4 rLP2086-B (B01 (SEQ ID NO: 58)) MnB non- rPA05 (SEQ ID NO: 55) Formulation Histidine buffer, pH 6.5 ted (L35408-140), Development, (L44130-129), Polysorbate tetravalent rPB44(SEQ ID NO: 44) Pearl River, 80 (L44130-127), L44857-50 (L37024-36A), rPB22 NY Trehalose (L44863-68), (SEQ ID NO: 75) WFI (B|Braun J0A012) (L37024-61), rPB09 (SEQ ID NO: 49)(L43930-80) AlPO4 0.5 mg/mL: L44863-86A Pfizer Pearl AlPO4 bulk H000000606- 0.25 mg/mL: L44863-86B River, NY D86864M 0.9% saline (B/Broun ), WFI (B/Broun J0A012) 60 mM Saline 962-UPD004 CSMD, Pfizer N/A Pearl River, Contain/Closure for MnB Tetravalent: Vials: 2 mL type-1 glass, West ceuticals rs: 13 mm vial stoppers for lyophilization, gray butyl, coated with Flurotec (WPS V2-F451W 4432/50 Gray B2-TR  RU Verisure Ready-Pack), West Pharmaceuticals Contain/Closure for 60 mM Saline: Vials: 2mL type-1 glass, Schott (Vendor Part #: 8M002PD-CS) Stoppers: 13 mm Daikyo D777-1, S2-F451, B2-40 Westar RS West, (Vendor Part #: 19560180) Container/Closure for AlPO4 Solutions: Vials: Sterile Empty Vials, Size 30 mL-20 mm, Stoppers included, Allergy Laboratories, Lot # SEV070708A TABLE 25. DATA ANALYSIS Table 25: Analytical Tests of MnB non-lipidated Antigen Lot L44857-50 Target A05 B44 B22 B09 B22, B09, Concentrati Concentrati Test Concentrati Concentrati A05, B44 on (µg/mL) on (µg/mL) on (μg/mL) on (μg/mL) (μg/mL) 60/60/60/6 64.1 63.0 LC 59.7 61.9 pH 6.5 6.52 Appearanc Clear, Lyo: White, fluffy cake. e colorless Reconstitution (w/ 60mM NaCl): Clear, colorless solution solution Moisture < 3% 0.60 % Lyophilized formulation was reconstitituted with Mobile Phase A during tation of B22, B09, A05, and B44 by IEX-HPLC; and with 60 mM NaCl diluent for pH and appearance.
Karl-Fischer (ICH) method was used to measure moisture (using methanol to reconstitute lyophilized formulations).
Table 26: pH and Appearance of AlPO4 Solutions Sample Lot # pH Appearance AlPO4 @ 0.5 mg/mL L44863-86A 5.95 Cloudy, white to off white suspension AlPO4 @ 0.25 L44863-86B 5.91 Cloudy, white to off mg/mL white suspension The non-lipidated tetravalent n (B22, B09, A05 and B44) were monitored for stability for 6 hours at 2-8 0C upon combination with MENVEO®.
Example 21: Serum Bactericidal Assay (SBA) A microcolony-based serum bactericidal assay (SBA) against multiple oup B, C and Y ococcal strains (Table 27) was performed on individual serum samples. Human sera from donors were qualified as the complement source for the strain tested in the assay. Complement-mediated antibody-dependent bactericidal titers were interpolated and expressed as the reciprocal of the dilution of the test serum that killed 50% of the meningococcal cells in the assay. The limit of detection of the assay was an SBA titer of 4. An SBA titer of <4 was assigned number of 2. A ≥ 4-fold rise of SBA titers in the week 10 sera in ison to the titers in the pre-bleed was calculated and compared.
Table 27 SBA Strains Serogroup fHBP Variant Strain name B A05 PMB1745 B B02 PMB17 B B09 PMB1489 B B16 PMB2882 B B44 PMB147 C A68 PMB2432 C B24 PMB2240 Y A121 PMB3386 Y B09 PMB3210 e 22: Immunogenicity of the combination of lipidated or non-lipidated factor H binding proteins and the conjugated vaccine in New Zealand white rabbits Serum bactericidal antibody is the immunologic surrogate of protection against meningococcal disease. Whether immunization with lipidated, non-lipidated rfHBP, tetravalent conjugate es alone or in ation elicited bactericidal antibodies in rabbits was determined by SBA. SBA measures the level of antibodies in a serum sample by mimicking the complement-mediated bacterial lysis that occurs naturally. In humans a SBA titer of 1:4 is considered the protective; a four fold rise in titer, pre vs post immunization also considered to be an immunologically relavant immune response.
Rabbit serum samples collected from weeks 0 and 10 were analyzed by SBA against strains of several meningococcal serogroups. As shown in Table 28 (higher dose) and 29 (lower dose), one week after the third immunization (week 10), the tetravalent conjugate vaccines only elicited SBA responses against MnC and MnY s tested.
All other serum samples yed bactericidal activity t the homologous strains as well as other test strains from the same fHBP subfamily as in the vaccine formulations. It is noted that zation with lipidated A05/B01 (SEQ ID NOs: 13 and 58, respectively) alone at 30 mcg dose each elicited the highest bactericidal antibodies against the gous strains as well as against other tested strains from both fHBP subfamilies (Table 28). Similarly, immunization with non -lipidated A05/B09/B22/B44 (SEQ ID NOs: 55, 49, 75, and 44, respectively) alone also elicited bactericidal antibodies against strains of several meningococcal serogroups, even though the SBA titers were 3 to der lower than the lipidated bivalent e (Table 30). A 100% responder rate (≥ 4-folder rise in an SBA titer) was achieved against all strains of various sergroups for lipidated fHBP, high dose of non-lipidated fHBP and all the combinations.
Table 28 Fold rise increase in SBA titers against meningococcus oup B, C and Y strains using sera from rabbits immunized with a higher dose ation of fHBPs and conjugate vaccine Fold Rise in PD3 SBA Titers MnB strains MnC MnY strains strains VACCINE Dose A05 B02 B09 B16 B44 A68 B24 A121 B09 MENVEO 1 hu 1 2 1 1 1 244 53 708 226 MENVEO/ 1 hu 349 871 279 806 2048 1592 401 1037 894 lipidated A05/B01 dose, proteins: mcg Lipidated A05/B01 30 mcg 591 624 745 842 1955 1926 344 595 905 Non-lipidated 30 mcg 39 105 192 300 391 61 137 52 148 A05/B09/B22/B44 each MENVEO/non- 1 hu 34 98 108 113 178 219 125 299 135 lipidated dose, A05/B09/B22/B44 proteins: mcg Rabbits pre-bleed sera showed no pre-existing bactericidal activity against the tested strains. NZW rabbits (n=3) were vaccinated at weeks 0, 4 and 8 with 0.5 mL vaccine, im; data Wk10 Table 29 Fold rise increase in SBA titers against meningococcus serogroup B, C and Y strains using sera from rabbits immunized with a lower dose combination of fHBPs and conjugate vaccine Fold Rise in PD3 SBA Titers MnB strains MnC MnY strains strains VACCINE Dose A05 B02 B09 B16 B44 A68 B24 A121 B09 MENVEO 1:10 hu 1 1 2 1 1 49 24 81 143 MENVEO/ lipidated 1:10 hu 191 140 124 336 926 940 172 560 366 1 dose, proteins: 3 mcg ted A05/B01 3 mcg 142 164 440 246 834 476 162 515 294 Non-lipidated 3 mcg 6 22 29 22 40 34 39 16 25 A05/B09/B22/B44 each MENVEO/non- 1:10 hu 10 52 76 60 158 102 100 122 lipidated dose, A05/B09/B22/B44 proteins: 3 mcg Rabbits pre-bleed sera showed no pre-existing bactericidal activity t the tested strains. NZW rabbits (n=3) were vaccinated at weeks 0, 4 and 8 with 0.5 mL vaccine, im; data Wk10 Table 30 SBA responder rates t meningococcus serogroup B, C and Y strains using sera from rabbits immunized with a ation of fHBPs and conjugate vaccine PD3 Responders (4 fold rise) MnB s MnC MnY strains strains VACCINE Dose A05 B02 B09 B16 B44 A68 B24 A121 B09 MENVEO 1 hu dose 0 0 0 0 0 100 100 100 100 MENVEO 1:10 hu 0 0 0 0 0 100 100 100 100 MENVEO/ lipidated 1 hu 100 100 100 100 100 100 100 100 100 A05/B01 dose, μg MENVEO/ lipidated 1:10 hu 100 100 100 100 100 100 100 100 100 A05/B01 dose, proteins: 3 μg each Lipidated A05/B01 30 μg 100 100 100 100 100 100 100 100 100 Lipidated A05/B01 3 μg each 100 100 100 100 100 100 100 100 100 Non-lipidated 30 μg 100 100 100 100 100 100 100 100 100 A05/B09/B22/B44 each Non-lipidated 3 μg each 67 67 67 67 100 67 100 67 100 A05/B09/B22/B44 MENVEO/non- 1 hu 100 100 100 100 100 100 100 100 100 lipidated dose, A05/B09/B22/B44 proteins: μg MENVEO/non- 1:10 hu 67 100 100 100 100 100 100 100 100 lipidated dose, A05/B09/B22/B44 proteins: 3 μg each NZQ rabbits (n=3) were vaccinated at weeks 0, 4 and 8 with 0.5 mL vaccine, im; data Lipidated fHBP elicited higher SBA titers than the non-lipidated fHBP.
The lipidated fHBP at 30 mcg each per dose elicited 3folder higher SBA titers to all the meningococcal B, C and Y strains tested. The pidated fHBP at 30 mcg each per dose elicited older higher SBA titers to all the meningococcal B, C and Y strains tested (Tables 28-29).
Dose ion was achieved with the fHBPs, the conjugate vaccine or the combinations With a higher dose of conjugate vaccine, fHBPs or their combinations increased the SBA responses than with a lower dose (Tables 28-30). The one human dose of the conjugate vaccine elicited 2folder high SBA titers against meningococcal C and Y strains than the one tenth dose of the ate vaccine. The lipidated fHBP at 30 mcg each per dose ed 2-4 folder high SBA titers against all the strains tested than the 3 mcg each per dose. The non-lipidated fHBP at 30 mcg each per dose elicited 4 folder high SBA titers against all the meningococcal serogroups B, C and Y strains than the 3 mcg each per dose.
Synergistic SBA responses by combination of fHBP and conjugate vaccines There is a trend that the SBA responses are higher against meningococcal serogroups C and Y strains when the combination of conjugate e and fHBP was used than by using either component alone, especially with the addition of a lower dose of ted or non-lipidated fHBP (Table 29). In the present study, the functional activity was evaluated against strains of several meningococcal serogroups using sera from New Zealand white rabbits immunized with recombinant lipidated or non-lipidated fHBP in formulation with AlPO4 and the conjugate vaccine alone or in combination. Rabbits receiving the conjugate vaccine elicited SBA responses only against meningococcal serogroup C and Y strains, but not to the serogroup B strains. The lipidated or idated fHBP in formulation with AlPO4 ed serum antibodies which were bactericidal against strains of all the meningococcal serogroups tested.
New Zealand white s receiving three doses of the ted or non-lipidated fHBP in formulation with AlPO4 elicited serum antibodies which were bactericidal against meningococcal serogroups B, C and Y strains tested. A 100% of responder rate (>4-folder rise in an SBA titer) was achieved against all the strains tested except the lower dose non-lipidated group.
The lipidated fHBP elicited greater bactericidal antibody titers than the nonlipidated forms. A clear dose response was observed with the lipidated or non-lipidated fHBP and the ate vaccine alone or in combinations.
There is a trend of istic SBA responses against meningococcal serogroup C and Y strains between the conjugate vaccine and fHBP especially at the addition of lower dose proteins.
Example 23: Evaluation of the immunogenicity of combinations of non-lipidated factor H binding proteins in New Zealand White Rabbits Studies were carried out in New Zealand White rabbits in the 2.5-3.5 kg range obtained from Charles River, Canada (Table 31). Rabbits were vaccinated intramuscularly at the hind leg, 0.5mL per site (1.0mL per dose, see Table 32) at weeks 0, 4 and 9. The Sequence ID Numbers for each of the antigens tested are listed in Table 33. There were 10 rabbits per group. Rabbits were bled at weeks 0, 6 and exsanguinated at week 10. Serum s were ed and week 0 and 10 serum samples were ed in the SBA against a panel of N. meningitidis isolates.
Table 31: Rabbits Used in these Studiesa Species Rabbit Strain New Zealand White Source Charles River Laboratory Number Animals per group 10 Sex Female Weight 2.5-3.5 kg a Rabbits were maintained in ance with established Institutional Animal Care and Use Committee guidelines Table 32: Study Designa # of rabbits Antigenic composition Lipidated Dose AlPO4 fHBP Variants (0.25mg/dose) A62 + B44 No 10mcg each Yes A05 + A62 + B44 No 10mcg each Yes A05 + A62 + B09 + B44 No 10mcg each Yes A05 + A62 +B09 + B44 No 5mcg each Yes A05 + A12 +B09 + B44 No 5mcg each Yes A12 + A62 + B09 + B44 No 5mcg each Yes A05 + A12 + A62 + B09 + B44 No 5mcg each Yes A05 + B01 Yes 10mcg each Yes a Rabbits were vaccinated intramuscularly (weeks 0, 4 and 9) and bled (weeks 0, 6 and ) to prepare serum s for SBA analysis Table 33: N. meningitidis Serogroup B fHBP Protein Variants Used rP2086-A05 SEQ ID NO: 13, wherein the Cys at position 1 is deleted, or SEQ ID NO: 55, e.g., encoded by SEQ ID NO: 54 rP2086-A12 SEQ ID NO: 14, wherein the Cys at position 1 is deleted, or SEQ ID NO: 66, e.g., encoded by SEQ ID NO: 67 rP2086-A62 SEQ ID NO: 70, wherein the Cys at position 1 is d, or SEQ ID NO: 71, e.g., encoded by SEQ ID NO: 72 rP2086-B09 SEQ ID NO: 18, wherein the Cys at on 1 is deleted, or SEQ ID NO: 49 rP2086-B44 SEQ ID NO: 21, wherein the Cys at position 1 is deleted, or SEQ ID NO: 44, e.g., encoded by SEQ ID NO: 43 6-A05 SEQ ID NO: 76 rLP2086-B01 SEQ ID NO: 58 Table 34 summarizes the immune response in rabbits to mixtures of idated fHBP proteins compared to the immune response to the rLP2086-A05 and rLP2086-B01 pair of lipidated antigens. Rabbit pre-bleed sera generally showed no preexisting icidal activity against the tested strains. The immune response is presented as the percent of animals in each treatment group that respond to the respective combinations of fHBP antigens following the third zation with an se in SBA titer of >4 fold. The SBA assay was performed using target N. meningitidis s that either express fHBP variants identical to the e immunogen (A05, A12), or strains that express heterologous fHBP variants (A22, B16, B24). The comparative amino acid sequence identity of the A22 fHBP variant diverges up to 15% from the subfamily A variants tested. Similarly, the comparative amino acid sequence identity of the B16 and B24 fHBP variants diverges up to 12% from the subfamily B variants included as antigens.
Table 34: Percent of New Zealand White Rabbits Vaccinated with Recombinant Non-lipidated fHBPs that d With a >4 Fold Rise in SBA Titers ose Three % Responders at PD3 with > 4X rise SBA Titers Immunogena Lipidated Dose per A05 A12 A22 B16 B24 antigen (mcg/0.5 A62 + B44 No 10 nd 50 100 100 50 A05 + A62 + B44 No 10 nd 40 80 80 60 A05 + A62 + B09 + B44 No 10 nd 60 100 100 100 A05 + A62 + B09 + B44 No 5 nd 40 40 100 70 A05 + A12 + B09 + B44 No 5 60 40 60 60 60 A12 + A62 + B09 + B44 No 5 100 70 100 100 70 A05 + A12 + A62 + B09 No 5 100 100 100 100 60 + B44 A05 + B01 Yes 10 nd 80 90 100 90 a 10 animals per treatment group; all treatments formulated with AlPO 4 adjuvant (250mcg/dose) In those groups of rabbits zed with 10mcg of each test rP2086 variant, serum samples from animals treated with the combination of A05 + A62 + B09 + B44 had the highest bactericidal response rate. The SBA response was somewhat reduced in animals treated with only 5mcg each of the same e of four non-lipidated fHBP variants. Other 4-valent groups of fHBP antigens dosed at 5mcg did as well as the combination of non-lipidated A05 + A62 + B09 + B44. Of the 4-valent combinations tested, serum samples from the treatment group that included 5mcg each of non- lipidated fHBP variants A12 + A62 + B09 + B44 had the best SBA response rates for the selected assay strains. The response rate to the pentavalent non-lipidated combination of A05 + A12 + A62 + B09 + B44 is somewhat better than the se to any of the 4-valent combinations tested.

Claims (22)

What we claim is:
1. An isolated polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 66.
2. The polypeptide according to claim 1, wherein the polypeptide is lipidated.
3. The polypeptide according to claim 1, wherein the polypeptide is non-lipidated.
4. The ptide according to claim 1, wherein the polypeptide is non-pyruvated.
5. The polypeptide according to claim 1, wherein the polypeptide is non-lipidated and non-pyruvated.
6. The polypeptide according to claim 1, wherein the ptide is immunogenic.
7. An immunogenic composition comprising the polypeptide as in any of claims 1 to
8. An isolated nucleic acid encoding an isolated polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 66.
9. The ed nucleic acid according to claim 8, wherein the nucleic acid sequence consists of SEQ ID NO: 67.
10. A method of ng an immune response against Neisseria meningitidis in a mammal excluding a human being comprising administering to the mammal an effective amount of an immunogenic composition sing an isolated polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 66.
11. A method of eliciting a bactericidal antibody t Neisseria meningitidis in a mammal excluding a human being comprising administering to the mammal an effective amount of an immunogenic composition comprising an isolated polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 66.
12. An genic composition comprising an isolated non-lipidated, nonpyruvylated ORF2086 polypeptide from Neisseria meningitidis serogroup B, and at least one conjugate selected from: a) a conjugate of a capsular saccharide of Neisseria meningitidis serogroup A, b) a conjugate of a capsular saccharide of ria meningitidis oup C, c) a ate of a capsular saccharide of Neisseria meningitidis serogroup W135; d) a conjugate of a capsular saccharide of Neisseria meningitidis serogroup Y, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO: 66.
13. The immunogenic composition according to claim 12, wherein the ition comprises at least two conjugates ed from: a) a conjugate of a capsular saccharide of Neisseria meningitidis serogroup A, b) a conjugate of a capsular ride of Neisseria meningitidis serogroup C, c) a conjugate of a ar saccharide of Neisseria meningitidis serogroup W135; d) a conjugate of a capsular ride of Neisseria meningitidis oup Y.
14. The immunogenic composition according to claim 12, wherein the composition comprises at least three conjugates selected from: a) a ate of a capsular saccharide of Neisseria meningitidis serogroup A, b) a ate of a capsular saccharide of Neisseria meningitidis serogroup C, and c) a conjugate of a capsular saccharide of Neisseria meningitidis serogroup W135; d) a conjugate of a capsular saccharide of Neisseria itidis serogroup Y.
15. The immunogenic composition according to claim 12, wherein the composition comprises a ate of a capsular ride of Neisseria meningitidis serogroup A; a ate of a capsular saccharide of Neisseria itidis serogroup C; a conjugate of a capsular saccharide of Neisseria meningitidis serogroup W135; and a conjugate of a ar saccharide of Neisseria meningitidis serogroup Y.
16. Use of an immunogenic composition according to any one of claims 7 or 12 to 15 in the manufacture of a medicament for inducing an immune response against ria meningitidis in a mammal.
17. Use of an immunogenic composition according to any one of claims 7 or 12 to 15 in the manufacture of a medicament for eliciting a bactericidal dy against Neisseria meningitidis in a mammal.
18. An isolated polypeptide accordingly to claim 1 substantially as herein described with reference to any example thereof and with or without reference to any one or more of the accompanying figures.
19. An immunogenic composition ing to claim 7 or 12 substantially as herein described with reference to any example thereof and with or without reference to any one or more of the accompanying figures.
20. An isolated nucleic acid according to claim 8 substantially as herein described with reference to any example thereof and with or without reference to any one or more of the accompanying figures.
21. A method according to claim 10 or 11 substantially as herein described with reference to any example thereof and with or without reference to any one or more of the accompanying figures.
22. Use according to claim 16 or 17 substantially as herein described with reference to any example thereof and with or without reference to any one or more of the accompanying figures. GTC GGCACA CC T GGAACACT GAC CCAAAAC 1) TT C 2) Sequences TC CAT ID NO: ID NO: Nucleic Acid TGGAAGAC Sequence (SEQ Sequence (SEQ CT GACAT GAAATC C Non-lipidated Variant c Acid Nucleic Acid >A04 t TGCAGCAGCGGAGGCGGAGGCGGCGGTGTCGCCGCCGACATCGGCACGGGGCTTGCCGATGCACTAACTGCGCCGCTCGACC ACAAAGGTTTGAAATCCCTGACATTGGAAGACTCCATTCCCCAAAACGGAACACTGACCCTGTCGGCACAAGGTGC GGAAAAAACTTTCAAAGCCGGCGACAAAGACAACAGCCTCAACACGGGCAAACTGAAGAACGACAAAATCAGCCGCTTCGAC TTCGTGCAAAAAATCGAAGTGGACGGACAAACCATCACACTGGCAAGCGGCGAATTTCAAATATACAAACAGGACCACTCCG CCGTCGTTGCCCTACAGATTGAAAAAATCAACAACCCCGACAAAATCGACAGCCTGATAAACCAACGCTCCTTCCTTGTCAG CGGTTTGGGCGGAGAACATACCGCCTTCAACCAACTGCCCGGCGACAAAGCCGAGTATCACGGCAAAGCATTCAGCTCCGAC GATGCCGGCGGAAAACTGACCTATACCATAGATTTTGCCGCCAAACAGGGACACGGCAAAATCGAACACCTGAAAACACCCG AGCAAAATGTCGAGCTTGCCGCCGCCGAACTCAAAGCAGATGAAAAATCACACGCCGTCATTTTGGGCGACACGCGCTACGG CAGCGAAGAAAAAGGCACTTACCACCTCGCCCTTTTCGGCGACCGCGCCCAAGAAATCGCCGGCTCGGCAACCGTGAAGATA GGGGAAAAGGTTCACGAAATCGGCATCGCCGGCAAACAGTAG P2086 >A05 Variant TGCAGCAGCGGAAGCGGAAGCGGAGGCGGCGGTGTCGCCGCCGACATCGGCACAGGGCTTGCCGATGCACTAACTGCGCCGC CATAAAGACAAAGGTT T TC GAC AGGTGCGGAAAAAACTTTCAAAGTCGGCGACAAAGACAACAGTCTCAATACAGGCAAATTGAAGAACGACAAAATCAGCCGC TTCGACTTTGTGCAAAAAATCGAAGTGGACGGACAAACCATCACGCTGGCAAGCGGCGAATTTCAAATATACAAACAGGACC ACTCCGCCGTCGTTGCCCTACAGATTGAAAAAATCAACAACCCCGACAAAATCGACAGCCTGATAAACCAACGCTCCTTCCT TGTCAGCGGTTTGGGCGGAGAACATACCGCCTTCAACCAACTGCCCAGCGGCAAAGCCGAGTATCACGGCAAAGCATTCAGC TCCGACGATGCCGGCGGAAAACTGACCTATACCATAGATTTTGCCGCCAAACAGGGACACGGCAAAATCGAACACCTGAAAA CACCCGAGCAGAATGTCGAGCTTGCCTCCGCCGAACTCAAAGCAGATGAAAAATCACACGCCGTCATTTTGGGCGACACGCG CTACGGCAGCGAAGAAAAAGGCACTTACCACCTCGCTCTTTTCGGCGACCGAGCCCAAGAAATCGCCGGCTCGGCAACCGTG AAGATAAGGGAAAAGGTTCACGAAATCGGCATCGCCGGCAAACAGTAG GGGCGGAGA GGGCGGAGA GGTT T GGTT T GTCAGC GTCAGC CT T CT T TT C TT C TC C TC C 3) CAACGC NO: 4) CAACGC ID NO: CT GATAAAC (SEQ ID CT GATAAAC ce (SEQ CGAGAGC Sequence CGACAGC CGAGAAAAT CGAGAAAAT Nucleic Acid TGCAGCAGCGGAGGCGGCGGTGTCGCCGCCGACATCGGCGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAG ACAAAAGTTTGCAGTCTTTGACGCTGGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAA TGGAAACGGCGACAGCCTCAATACGGGCAAATTGAAGAACGACAAGGTCAGCCGCTTCGACTTTATCCGTCAAATC GAAGTGGACGGACAAACCATCACGCTGGCAAGCGGCGAATTTCAAATATACAAACAGAACCACTCCGCCGTCGTTGCCCTAC CAACAACC C Nucleic Acid CAACAACC C >A12 Variant AGATT AT ACATACCGCCTTCAACCAACTGCCTGACGGCAAAGCCGAGTATCACGGCAAAGCATTCAGCTCCGACGACCCGAACGGCAGG CTGCACTACTCCATTGATTTTACCAAAAAACAGGGTTACGGCAGAATCGAACACCTGAAAACGCCCGAGCAGAATGTCGAGC TTGCCTCCGCCGAACTCAAAGCAGATGAAAAATCACACGCCGTCATTTTGGGCGACACGCGCTACGGCGGCGAAGAAAAAGG CACTTACCACCTCGCCCTTTTCGGCGACCGCGCCCAAGAAATCGCCGGCTCGGCAACCGTGAAGATAAGGGAAAAGGTTCAC GAAATCGGCATCGCCGGCAAACAGTAG >A12-2 Variant TGCAGCAGCGGAGGGGGCGGTGTCGCCGCCGACATTGGTGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAG ACAAAAGTTTGCAGTCTTTGACGCTGGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAA AACTTATGGAAACGGCGACAGCCTCAATACGGGCAAATTGAAGAACGACAAGGTCAGCCGCTTCGACTTTATCCGTCAAATC GAAGTGGACGGACAAACCATCACGCTGGCAAGCGGCGAATTTCAAATATACAAACAGAACCACTCCGCCGTCGTTGCCCTAC AGATT GAAAAAAT ACATACCGCCTTCAACCAACTGCCTGACGGCAAAGCCGAGTATCACGGCAAAGCATTCAGCTCCGACGACCCGAACGGCAGG CTGCACTACTCCATTGATTTTACCAAAAAACAGGGTTACGGCAGAATCGAACACCTGAAAACGCCCGAGCAGAATGTCGAGC TTGCCTCCGCCGAACTCAAAGCAGATGAAAAATCACACGCCGTCATTTTGGGCGACACGCGCTACGGCGGCGAAGAAAAAGG CACTTACCACCTCGCCCTTTTCGGCGACCGCGCCCAAGAAATCGCCGGCTCGGCAACCGTGAAGATAAGGGAAAAGGTTCAC GAAATCGGCATCGCCGGCAAACAGTAG GGGTGGAGA CCTGTCGGC GGTT T TGAG GACAAAATCAGC GTCAGC CT T TT C TC C CCAAAACGGAACAC TT C CAACACAGGCAAACT GAAGAAC 5) CAACGC TC CAT ID NO: NO: 6) TGGAAGAC CT GATAAAC Sequence (SEQ AGCGGAGGCGGCGGTGTCGCCGCCGACATCGGCGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAG ACAAAAGTTTGCAGTCTTTGACGCTGGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAA AACTTATGGAAACGGCGACAGCCTCAATACGGGCAAATTGAAGAACGACAAGGTCAGCCGCTTCGACTTTATCCGTCAAATC GAAGTGGACGGGCAGCTCATTACCTTGGAGAGCGGAGAGTTCCAAATATACAAACAGGACCACTCCGCCGTCGTTGCCCTAC (SEQ ID CGAGAGC Sequence CT GAGAT GAAATC C GACAAAGACAACAGTC T CGGC Nucleic Acid CAACAACC C Nucleic Acid GACAAAGGTT T TCAAAGC GGAAAGAACT T AGATT AT ACATACCGCCTTCAACCAACTGCCCAGCGGCAAAGCCGAGTATCACGGCAAAGCATTCAGCTCCGACGATGCTGGCGGAAAA CTGACCTATACCATAGATTTCGCCGCCAAACAGGGACACGGCAAAATCGAACACTTGAAAACACCCGAGCAAAATGTCGAGC TTGCCTCCGCCGAACTCAAAGCAGATGAAAAATCACACGCCGTCATTTTGGGCGACACGCGCTACGGCGGCGAAGAAAAAGG CACTTACCACCTCGCCCTTTTCGGCGACCGCGCCCAAGAAATCGCCGGCTCGGCAACCGTGAAGATAAGGGAAAAGGTTCAC GAAATCGGCATCGCCGGCAAACAGTAG >B02 Variant TGCAGCAGCGGAGGCGGCGGAAGCGGAGGCGGCGGTGTCGCCGCCGACATCGGCGCGGGGCTTGCCGATGCACTAACCGCAC TC GAG CGC >A22 Variant ACAAGGTGC CGCTTCGACTTTATCCGTCAAATCGAAGTGGACGGGCAGCTCATTACCTTGGAGAGCGGAGAGTTCCAAGTGTACAAACAAA GCCATTCCGCCTTAACCGCCCTTCAGACCGAGCAAGTACAAGACTCGGAGCATTCCGGGAAGATGGTTGCGAAACGCCAGTT CAGAATCGGCGACATAGTGGGCGAACATACATCTTTTGACAAGCTTCCCAAAGACGTCATGGCGACATATCGCGGGACGGCG TTCGGTTCAGACGATGCCGGCGGAAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGACACGGCAAAATCGAACATT TGAAATCGCCTGAACTCAATGTTGACCTGGCCGCCGCCGATATCAAGCCGGATGAAAAACACCATGCCGTCATCAGCGGTTC CGTCCTTTACAACCAAGCCGAGAAAGGCAGTTACTCTCTAGGCATCTTTGGCGGGCAAGCCCAGGAAGTTGCCGGCAGCGCG GAAGTGGAAACCGCAAACGGCATACGCCATATCGGTCTTGCCGCCAAGCAATAA 7) ID NO: NO: 8) Sequence (SEQ (SEQ ID Sequence Nucleic Acid Nucleic Acid >B03 Variant TGCAGCAGCGGAGGCGGCGGTGTCGCCGCCGACATCGGCGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAG ACAAAAGTTTGCAGTCTTTGACGCTGGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAA TGGAAACGGCGACAGCCTTAATACGGGCAAATTGAAGAACGACAAGGTCAGCCGTTTCGACTTTATCCGTCAAATC GAAGTGGACGGGCAGCTCATTACCTTGGAGAGCGGAGAGTTCCAAGTGTACAAACAAAGCCATTCCGCCTTAACCGCCCTTC AGACCGAGCAAGAACAAGATCCAGAGCATTCCGGGAAGATGGTTGCGAAACGCCGGTTCAAAATCGGCGACATAGCGGGCGA ACATACATCTTTTGACAAGCTTCCCAAAGACGTCATGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCCGGCGGA AAACTGACCTATACTATAGATTTTGCTGCCAAACAGGGACACGGCAAAATCGAACATTTGAAATCGCCCGAACTCAATGTCG AGCTTGCCACCGCCTATATCAAGCCGGATGAAAAACACCATGCCGTCATCAGCGGTTCCGTCCTTTACAATCAAGACGAGAA AGGCAGTTACTCCCTCGGTATCTTTGGCGGGCAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTGGAAACCGCAAACGGCATA CACCATATCGGTCTTGCCGCCAAGCAATAA >B09 Variant TGCAGCAGCGGAGGGGGCGGTGTCGCCGCCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAG ACAAAGGTTTGCAGTCTTTAACGCTGGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAA AACTTATGGAAACGGCGACAGCCTTAATACGGGCAAATTGAAGAACGACAAGGTCAGCCGCTTCGACTTTATCCGTCAAATC GAAGTGGACGGGAAGCTCATTACCTTGGAGAGCGGAGAGTTCCAAGTGTACAAACAAAGCCATTCCGCCTTAACCGCCCTTC AGCAAGTACAAGACTCGGAGGATTCCGGGAAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGCGA ACATACATCTTTTGACAAGCTTCCCAAAGGCGGCAGTGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCTGGCGGA AAACTGACCTATACTATAGATTTCGCCGCCAAGCAGGGACACGGCAAAATCGAACATTTGAAATCGCCCGAACTCAATGTCG AGCTTGCCACCGCCTATATCAAGCCGGATGAAAAACGCCATGCCGTTATCAGCGGTTCCGTCCTTTACAACCAAGACGAGAA AGGCAGTTACTCCCTCGGTATCTTTGGCGGGCAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTGGAAACCGCAAACGGCATA CACCATATCGGTCTTGCCGCCAAGCAGTAA 9) ID NO: NO: 10) Sequence (SEQ (SEQ ID Sequence Nucleic Acid Nucleic Acid >B22 Variant TGCAGCAGCGGAGGCGGCGGTGTCGCCGCCGACATCGGCGCGGTGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAG ACAAAAGTTTGCAGTCTTTGACGCTGGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAA AACTTATGGAAACGGCGACAGCCTCAATACGGGCAAATTGAAGAACGACAAGGTCAGCCGCTTCGACTTTATCCGTCAAATC GAAGTGGACGGGCAGCTCATTACCTTGGAGAGCGGAGAGTTCCAAGTGTACAAACAAAGCCATTCCGCCTTAACCGCCCTTC AGACCGAGCAAGTACAAGATTCGGAGCATTCAGGGAAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGATATAGCGGGTGA ATCTTTTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCATTCGGTTCAGACGATGCCAGTGGA AAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGACACGGCAAAATCGAACATTTGAAATCGCCAGAACTCAATGTTG ACCTGGCCGCCTCCGATATCAAGCCGGATAAAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTACAACCAAGCCGAGAA AGGCAGTTACTCTCTAGGCATCTTTGGCGGGCAAGCCCAGGAAGTTGCCGGCAGCGCAGAAGTGGAAACCGCAAACGGCATA CGCCATATCGGTCTTGCCGCCAAGCAGTAA >B24 Variant TGCAGCAGCGGAGGGGGTGGTGTCGCCGCCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAG ACAAAGGTTTGCAGTCTTTGACGCTGGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAA AACTTATGGAAACGGTGACAGCCTCAATACGGGCAAATTGAAGAACGACAAGGTCAGCCGTTTCGACTTTATCCGCCAAATC GAAGTGGACGGGCAGCTCATTACCTTGGAGAGTGGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTC AGACCGAGCAAATACAAGATTCGGAGCATTCCGGGAAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGCGA ATCTTTTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCCGGCGGA AAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAACTCAATGTCG ACCTGGCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTACAACCAAGCCGAGAA AGGCAGTTACTCCCTCGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTGAAAACCGTAAACGGCATA CGCCATATCGGCCTTGCCGCCAAGCAATAA CCTGTCGGC TGAG GACAAAATCAGC CCAAAACGGAACAC TT C CAACACAGGCAAACT C 11) TC CAT ID NO: TGGAAGAC Sequence (SEQ CT GAGAT GAAATC C GACAAAGACAACAGTC T CGGC Nucleic Acid TGCAGCAGCGGAGGCGGCGGAAGCGGAGGCGGCGGTGTCGCCGCCGACATCGGCGCGGGGCTTGCCGATGCACTAACCGCAC CATAAAGACAAAGGTT T TT CAAAGC GGAAAGAACT >B44 Variant TC GAG CGC ACAAGGTGC CGCTTCGACTTTATCCGTCAAATCGAAGTGGACGGGCAGCTCATTACCTTGGAGAGCGGAGAGTTCCAAGTGTACAAACAAA GCCATTCCGCCTTAACCGCCCTTCAGACCGAGCAAGTACAAGACTCGGAGCATTCCGGGAAGATGGTTGCGAAACGCCAGTT CAGAATCGGCGACATAGTGGGCGAACATACATCTTTTGGCAAGCTTCCCAAAGACGTCATGGCGACATATCGCGGGACGGCG TTCGGTTCAGACGATGCCGGCGGAAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGACACGGCAAAATCGAACATT CGCCAGAACTCAATGTTGACCTGGCCGCCGCCGATATCAAGCCGGATGAAAAACACCATGCCGTCATCAGCGGTTC CGTCCTTTACAACCAAGCCGAGAAAGGCAGTTACTCTCTAGGCATCTTTGGCGGGCAAGCCCAGGAAGTTGCCGGCAGCGCG GAAGTGGAAACCGCAAACGGCATACGCCATATCGGTCTTGCCGCCAAGCAATAA 12) 13) 14) 15) Acid Sequences ID NO: ID NO: ID NO: ID NO: Variant Amino Sequence (SEQ Sequence (SEQ Sequence (SEQ Sequence (SEQ Amino Acid Amino Acid Amino Acid Amino Acid P2086 Non-lipidated >A04 Variant CSSGGGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDSIPQNGTLTLSAQGAEKTFKAGDKDNSLNTGKLKNDKISRFD FVQKIEVDGQTITLASGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPGDKAEYHGKAFSSD DAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKI GEKVHEIGIAGKQ >A05 Variant CSSGSGSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAEKTFKVGDKDNSLNTGKLKNDKISR FDFVQKIEVDGQTITLASGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPSGKAEYHGKAFS SDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELASAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATV KIREKVHEIGIAGKQ >A12 Variant CSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQI ITLASGEFQIYKQNHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDPNGR LHYSIDFTKKQGYGRIEHLKTPEQNVELASAELKADEKSHAVILGDTRYGGEEKGTYHLALFGDRAQEIAGSATVKIREKVH EIGIAGKQ >A22 t CSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQI EVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPSGKAEYHGKAFSSDDAGGK LTYTIDFAAKQGHGKIEHLKTPEQNVELASAELKADEKSHAVILGDTRYGGEEKGTYHLALFGDRAQEIAGSATVKIREKVH EIGIAGKQ 16) 17) 18) 19) ID NO: ID NO: ID NO: ID NO: Sequence (SEQ ce (SEQ Sequence (SEQ Sequence (SEQ Amino Acid Amino Acid Amino Acid Amino Acid >B02 Variant CSSGGGGSGGGGVAADIGAGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAERTFKAGDKDNSLNTGKLKNDKIS RFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQTEQVQDSEHSGKMVAKRQFRIGDIVGEHTSFDKLPKDVMATYRGTA FGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAAADIKPDEKHHAVISGSVLYNQAEKGSYSLGIFGGQAQEVAGSA EVETANGIRHIGLAAKQ >B03 Variant CSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQI EVDGQLITLESGEFQVYKQSHSALTALQTEQEQDPEHSGKMVAKRRFKIGDIAGEHTSFDKLPKDVMATYRGTAFGSDDAGG KLTYTIDFAAKQGHGKIEHLKSPELNVELATAYIKPDEKHHAVISGSVLYNQDEKGSYSLGIFGGQAQEVAGSAEVETANGI HHIGLAAKQ >B09 Variant CSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQI EVDGKLITLESGEFQVYKQSHSALTALQTEQVQDSEDSGKMVAKRQFRIGDIAGEHTSFDKLPKGGSATYRGTAFGSDDAGG DFAAKQGHGKIEHLKSPELNVELATAYIKPDEKRHAVISGSVLYNQDEKGSYSLGIFGGQAQEVAGSAEVETANGI HHIGLAAKQ >B22 Variant CSSGGGGVAADIGAVLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQI EVDGQLITLESGEFQVYKQSHSALTALQTEQVQDSEHSGKMVAKRQFRIGDIAGEHTSFDKLPEGGRATYRGTAFGSDDASG KLTYTIDFAAKQGHGKIEHLKSPELNVDLAASDIKPDKKRHAVISGSVLYNQAEKGSYSLGIFGGQAQEVAGSAEVETANGI RHIGLAAKQ 20) 21) ID NO: ID NO: Sequence (SEQ Sequence (SEQ Amino Acid Amino Acid >B24 Variant GVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQI EVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKRQFRIGDIAGEHTSFDKLPEGGRATYRGTAFGSDDAGG KLTYTIDFAAKQGNGKIEHLKSPELNVDLAAADIKPDGKRHAVISGSVLYNQAEKGSYSLGIFGGKAQEVAGSAEVKTVNGI RHIGLAAKQ >B44 Variant CSSGGGGSGGGGVAADIGAGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAERTFKAGDKDNSLNTGKLKNDKIS RFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQTEQVQDSEHSGKMVAKRQFRIGDIVGEHTSFGKLPKDVMATYRGTA FGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAAADIKPDEKHHAVISGSVLYNQAEKGSYSLGIFGGQAQEVAGSA EVETANGIRHIGLAAKQ
NZ747917A 2012-03-09 2013-03-06 Neisseria meningitidis compositions and methods thereof NZ747917B2 (en)

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