US20130011429A1 - Immunogenic composition - Google Patents

Immunogenic composition Download PDF

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US20130011429A1
US20130011429A1 US13/583,163 US201113583163A US2013011429A1 US 20130011429 A1 US20130011429 A1 US 20130011429A1 US 201113583163 A US201113583163 A US 201113583163A US 2013011429 A1 US2013011429 A1 US 2013011429A1
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fhbp
antigen
suitably
protein
disclosure
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Jan Poolman
Cindy Castado
Vincent Weynants
Nathalie Isabelle Devos
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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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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)
    • 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
    • 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
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the disclosure relates to the field of Neisserial immunogenic compositions and vaccines, their manufacture and the use of such compositions in medicine.
  • Neisserial strains of bacteria are the causative agents for a number of human pathologies, against which there is a need for effective vaccines to be developed.
  • Neisseria gonorrhoeae and Neisseria meningitidis cause pathologies which could be treated by vaccination.
  • Neisseria meningitidis is an important pathogen, particularly in children and young adults. Septicemia and meningitis are the most life-threatening forms of invasive meningococcal disease (IMD). This disease has become a worldwide health problem because of its high morbidity and mortality.
  • IMD invasive meningococcal disease
  • Vaccines based on the capsular polysaccharide of serogroups A, C, W and Y have been developed and have been shown to control outbreaks of meningococcal disease (Peltola et al 1985 Pediatrics 76; 91-96).
  • serogroup B is poorly immunogenic and induces only a transient antibody response of a predominantly IgM isotype (Ala'Aldeen D and Cartwright K 1996, J. Infect. 33; 153-157).
  • the development of a vaccine against serogroup B meningococcus presents particular difficulties because the polysaccharide capsule is poorly immunogenic owing to its immunologic similarity to human neural cell adhesion molecule.
  • fHbp One antigen contemplated for use in vaccines against Neisseria meningitidis is fHbp.
  • Lewis et al discloses the status of fHbp as a vaccine candidate Expert Reviews Vaccines 8(6)p729, (2009).
  • an immunogenic composition comprising:
  • the present disclosure relates to a method of treatment or prevention of Neisserial infection or disease comprising administering to an individual in need thereof a protective dose of an immunogenic composition comprising:
  • an immunogenic composition comprising
  • kits comprising
  • the disclosure relates to a method for manufacture of an fHbp based composition for prevention or amelioration of Neisseria meningitidis infection or disease, the method comprising combining an fHbp antigen with a second antigen capable of generating an antibody response against a Neisseria meningitidis L2 immunotype.
  • FIG. 1 Level of expression of fHBP, by Western-Blot. Shows whole-cells expressing different level of fHBP: high (line 1), intermediate (line 7), low (line 5) and non-detectable (lines 2, 3, 4, 6)
  • FIG. 2 Alignment of mature fHbp protein sequences.
  • fHbp variant B of MC58 strain and fHBP variant A of 2996 strain are used as reference sequences.
  • Mature fHBP amino acid sequences were compared with the ClustalW method (MegAlign SoftWare from DNASTAR Lasergene 7).
  • FIG. 3 Tdfl structure
  • SEQ ID NO: 1 amino acid residues 1 to 137 of the mature sequence of a family B fHbp protein
  • SEQ ID NO: 2 amino acid residues 136 to 254 of the mature sequence of a family A fHbp protein
  • SEQ ID NO: 3 Consensus sequence from Family A over region 113-135
  • SEQ ID NO: 4 consensus sequence from Family B over region 113-135
  • SEQ ID NO: 6 nucleic acid sequence for mature Family B fHbp sequence from strain MC58
  • SEQ ID NO: 7 mature Family A fHbp sequence from strain 8047
  • SEQ ID NO: 8 nucleic acid sequence for mature Family A fHbp sequence from strain 8047
  • SEQ ID NO: 9 amino acid 27 to 273 of full length fHbp sequence from strain 8047 with histidine tag (LVL489)
  • SEQ ID NO: 10 nucleic acid sequence for LVL489
  • SEQ ID NO: 11 amino acid 73 to 320 of full length fHbp sequence from strain MC58 with histidine tag (LVL 490)
  • SEQ ID NO: 12 nucleic acid sequence for LVL490
  • SEQ ID NO: 13 amino acid 66-72 of the full length Family B fHbp sequence
  • SEQ ID NO: 14 sequence of Histidine affinity tag
  • SEQ ID NO: 15 The peptide GENT (aa residues 136-139 in 8047 of family A and MC 58 of family B mature sequences) identical in family A and B
  • SEQ ID NO: 16 amino acid sequence Fusion protein LVL491
  • SEQ ID NO: 17 nucleic acid sequence for LVL491
  • SEQ ID NO: 18 amino acid sequence of fusion protein A
  • SEQ ID NO: 19 nucleic acid sequence for fusion protein A
  • SEQ ID NO: 20 amino acid sequence of fusion protein B
  • SEQ ID NO: 21 nucleic acid sequence for fusion protein B
  • SEQ ID NO: 22 amino acid sequence of fusion protein C
  • SEQ ID NO: 23 nucleic acid sequence of fusion protein C
  • SEQ ID NO: 24 amino acid sequence of fusion protein E
  • SEQ ID NO: 25 nucleic acid sequence of fusion protein E
  • SEQ ID NO: 26 amino acids within positions 242-246 indicating Family A
  • SEQ ID NO: 27 amino acids within positions 242-246 indicating Family B
  • Neisserial meningitidis L3 immunotypes produce fHbp at a significantly lower level than strains expressing an L3 inner core LOS. See the comparison studies herein which revealed that 6/31 (19%) of L3 strains expressed low or undetectable fHbp, whereas 14/15 (93%) of L2 strains expressed low or undetectable fHbp.
  • a vaccine based upon fHbp alone will not be effective against all menB strains, and fHbp should be supplemented with a second antigen capable of generating an antibody response against a Neisseria meningitidis L2 immunotype.
  • the immunogenic compositions of the disclosure are suitably effective as a vaccine against Neisseria meningitides infection or disease.
  • the present disclosure relates to an immunogenic composition
  • an fHbp polypeptide such as fHbp A or B, or a combination of fHbp polypeptides, or chimeric fHbp.
  • this is a full length fHBP polypeptide or fragment thereof, which is capable of eliciting antibodies against fHbp.
  • fHbp may be a fusion polypeptide as disclosed in, for example, WO/2009/114485.
  • the fHbp may be from any fHbp family.
  • an fHbp polypeptide is any polypeptide that comprises an epitope capable of eliciting antibodies against fHbp.
  • fHbp is also referred to as as GNA1870 and lipoprotein 2086.
  • the fHbp polypeptide is a fusion protein.
  • the present disclosure identifies 2 families of fHbp protein, A and B, and thus allows for the construction of chimaeric fHbp fusion proteins with sequences from 2 families, providing protection against Neisseria meningitidis infection or disease.
  • chimaeric fHbp protein and fHbp fusion protein are used interchangeably herein.
  • the disclosure relates to a fusion protein comprising amino acid sequences F1 and F2, where F1 is a N-terminal fragment of a first fHbp amino acid sequence and F2 is a C-terminal fragment of a second fHbp amino acid sequence.
  • the fusion protein comprises an F1 N-terminal fragment from fHbp Family B and F2 C-terminal fragment from fHbp Family A.
  • F1 and F2 are at least 10 amino acids in length but suitably comprise 20 amino acids, more suitably 30 amino acids, more suitably 40 amino acids, suitably 50 amino acids, suitably 60 amino acids, suitably 70 amino acids, suitably 80 amino acids, suitably 90 amino acids, suitably 100 amino acids, suitably 110 amino acids or suitably 120 amino acids, suitably taken contiguously from the amino acid sequence of an fHbp protein.
  • the F1 and F2 fragments do not comprise overlapping sequences, when considered in respect of an alignment between the first and second fHbp family sequences.
  • the fusion protein F1+F2 has a combined length of at least 200 amino acids, suitably at least 210, at least 220, at least 230, at least 240, at least 250 amino acids, and in one aspect is a full length FHBP protein of 254 amino acids.
  • the 2 parts, F1 and F2, of any fusion protein of the disclosure do not need to be directly linked, and may include a linker between F1 and F2. However, in one aspect, the F1 and F2 parts are directly linked.
  • the fusion protein may have additions, substitutions or deletions.
  • both the F1 and F2 portions of the sequence comprise an immunogenic epitope.
  • the F1 or F2 portion of the sequence contains epitopes suitable for generation of antibodies against family A and family B fHbp proteins.
  • an N terminal fragment may be equivalent to, or part of, the 140 N-terminal amino acids of the mature sequence of a fHbp family B protein, suitably all or part of the N-terminal 135, 136, 137, 138 or 139 amino acids of the mature sequence of the fHbp protein.
  • Amino acid residues 1 to 137 of the mature sequence of a family B fHbp protein represent amino acid residues 66 to 202 of the full length family B fHbp sequence.
  • Suitable fragments are amino acid residues, 1 to 135, 1 to 136, 1 to 137, 1 to 138, 1 to 139, 8 to 135, 8 to 137 or 8 to 139 of the mature sequence of a family B fHbp protein.
  • Residues 1 to 137 of the mature sequence are represented by strain MC58 of Family B shown in Seq ID No. 1 below or the equivalent regions of other strains of Family B.
  • SEQ ID No. 1 CSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSL NTGKLKNKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKRQF RIGDIAGE
  • one or more of the first seven amino acids of the mature sequence of the F1 N terminal fragment are replaced by a histidine tag, or other affinity tag, to facilitate purification.
  • a histidine tag, or other affinity tag is added to the N terminus of the mature protein to facilitate purification.
  • a C-terminal fragment is equivalent to, or part of, amino acids 130-254 of the mature fHbp sequence of a family A fHbp protein, suitably all or part of amino acids 136-254, 137-254, 138-254, 139-254, or 140 to 254 of the mature sequence.
  • Amino acid residues 136 to 254 of the mature sequence of a family A fHbp protein represent amino acid residues 155 to 273 of the full length family A fHbp sequence.
  • Residues 136 to 254 of the mature sequence are represented by strain 8047 of Family A shown in Seq ID No. 2 below or the equivalent regions of other strains of Family A.
  • Amino acid residues GENT are conserved among family A and B (aa residues 136-139 in 8047 of family A and MC 58 of family B) and can therefore be included in the amino acid sequence from either family.
  • FHbp proteins are defined into two families, A and B, herein.
  • the family classification is disclosed in “Sequence Diversity of the Factor H Binding Protein Vaccine Candidate in Epidemiologically Relevant Strains of Serogroup B Neisseria meningitides . The Journal of infectious diseases 2009, vol. 200, no 3, pp. 379-389”
  • the family identity is assessed over region 136-254.
  • proteins in the same family have >80% identity based upon the sequence of fHbp starting from amino acid 136 of the mature protein to the C terminus.
  • proteins in different families have 50-75% identity based upon the sequence of fHbp starting from amino acid 136 of the mature protein to the C terminus.
  • the family identity is assessed over region 113-135.
  • proteins in the same family have >69% identity based upon the region 113-135 of the mature amino acid sequence of fHbp.
  • proteins in different families have ⁇ 20% identity based upon the region 113-135 of the mature amino acid sequence of fHbp.
  • Family A and B may be distinguished by the presence of one or more of the following amino acids:
  • family A and B comprises the following consensus sequence from region 113-135:
  • strain MC58 An example of a family B sequence (SEQ ID NO. 5) is strain MC58:
  • Amino acids identified within this sequence as being of potential importance include:
  • family B species include strains H44/76, M982, M060240006, 03s-0408, and other examples will be well known to the skilled person.
  • SEQ ID NO. 7 An example of a family A sequence (SEQ ID NO. 7) is strain 8047:
  • Amino acids identified within this sequence as being of potential importance include:
  • family A species include strains M1239, M981, M08 — 240117, M97252153, and other examples will be well known to the skilled person.
  • the fusion protein is suitably capable of eliciting antibodies against both family members A and B as defined herein.
  • the fusion protein is able to elicit neutralizing antibodies, suitably in response to infection by N. meningitidis expressing family A or family B fHbp molecules.
  • the chimaeric protein when administered at an effective dose, elicits a protective immune response against Neisserial infection, more suitably protective against N. meningitidis serogroup B infection.
  • the fusion protein is immunologically reactive with antibodies generated against Neisserial full-length fHbp proteins or with antibodies generated by infection of a mammalian host with Neisseria.
  • chimeric proteins are able to elicit the production of bactericidal antibodies mediating the complement killing of strains expressing either the fHbp A or fHbp B.
  • the fusion protein of the disclosure has at least one at least one mutation to prevent or reduce Factor H binding.
  • Factor H binding may be human factor H binding, for example as assessed by ELISA or surface plasmon resonance, as disclosed in M. C. Schneider et al., 2009 “Neisseria meningitidis recruits factor H using protein mimicry of host carbohydrates” Nature Letters.
  • the mutation to prevent factor H binding is contained within the C-terminal F2 fragment, from amino acids 136-254 of fHbp.
  • the mutation to prevent factor H binding comprises substituting at least one Glu to Ala in fHbp. In a further aspect the mutation to prevent factor H binding comprises substituting one or more of the following residues contained within an F2 fragment to prevent fHbp binding: Glu 217, Glu/Thr 238 of fHbp Family A; Glu 218, Glu 239 of fHbp Family B. In one aspect one or more of the residues are mutated to alanine.
  • Factor H binding may be assessed by ELISA.
  • fHbp poylpeptides (chimeric or not) may be coated on a microplate. After saturation and washes, purified human fH or recombinant human fH is incubated in microwells and binding to fHBP is revealed (after washes) via addition of rabbit antibodies directed against the human fH and subsequent incubation with anti-rabbit IgG conjugated to peroxidise.
  • the F1 fragment comprises both Gly at position 121 and Lys at position 122, (numbering based on the MC58 strain as a reference family B strain).
  • the fusion protein is capable of binding to antibodies JAR3 or JAR5
  • the F2 fragment comprises one, or more or all of the following amino acids (numbering based on the 8047 strain as a reference A strain): Ala 173; Ser 215; Lys 179 and Glu 191, and in one aspect comprises both Lys 179 and Glu 191.
  • the fusion protein is capable of binding to one or more of antibodies JAR10, JAR11, JAR13.
  • the F2 fragment is from family A
  • the fusion protein being constructed to comprise one or more or all of the following amino acids replacing the naturally occurring amino acids: ala217, optionally ala at position 238, Glu146, Gly inserted at position 146, after the glutamine (subsequent numbers being shifted by +1 with respect to the wild type 8047 sequence), Gly148, Arg149 and Arg204 (numbering based on the MC58 strain as a reference family B strain).
  • the fusion protein is capable of binding to MAb502
  • the fragment may comprises one or more or all of pro 145, phe 227, gly 228, lys 230 and glu 233.
  • the fusion protein is capable of binding to MAb502.
  • fHbp includes amino acids disclosed as being relevant for immunogenicity in any such publication.
  • the chimaeric protein of the disclosure comprising one or more amino acid alterations as defined above, demonstrates an increase of the bactericidal titers against strains expressing fHbp compared to the chimeric proteins without these modifications.
  • the fusion protein of the present disclosure comprises residues 1 to 135, 1 to 136, 1 to 137, 1 to 138 or 1 to 139 from a mature family B fHbp protein and residues 136 to 254, 137 to 254, 138 to 254, 139 to 254 or 140 to 254 of a mature family A fHbp protein, and is unable to bind to Factor H.
  • the fusion protein of the present disclosure comprises residues 8 to 135, 8 to 136, 8 to 137, 8 to 138 or 8 to 139 from a mature family B fHbp protein, optionally with a histidine tag, and residues 136 to 254, 137 to 254, 138 to 254, 139 to 254 or 140 to 254 of a mature family A fHbp protein, and is unable to bind to Factor H.
  • one or more of the first seven amino acids from a mature family B fHbp protein are absent and may be replaced by a histidine tag, or other affinity tag, to facilitate purification.
  • the family B fHbp portion of the fusion protein starts at residue 2, 3, 4, 5, 6 or 7 of the mature sequence.
  • the fusion polypeptide comprises residues 1 to 135, 1 to 136, 1 to 137, 1 to 138, or 1 to 139 from a family B fHbp protein, for example having the MC58 sequence, and residues 136 to 254, 137 to 254, 138 to 254, 139 to 254 or 140 to 254 of a family A fHbp protein, for example having the sequence of strain 8047, the fusion protein being constructed to comprise the following amino acids replacing the naturally occurring amino acids: Ala217, optionally Ala at position 238, Glu146, Gly inserted at position 146, after the glutamine (subsequent numbers being shifted by +1 with respect to the wild type 8047 sequence), Gly148, Arg149 and Arg204 of family B mature protein sequence from strain MC58.
  • the fusion polypeptide additionally comprises one or more or all of pro 145, phe 227, gly 228, lys 230 and glu 233. These amino acids are found in construct E exemplified below.
  • the fusion polypeptide is selected from fusion proteins LVL491 (SEQ ID NO. 16), A (SEQ ID NO. 18), B (SEQ ID NO. 20), C (SEQ ID NO. 22), D, E (SEQ ID NO. 24) or F as disclosed herein, particularly from fusion proteins A, B, C, and E.
  • the composition also comprises a second antigen capable of generating an antibody response against a Neisseria meningitidis L2 immunotype.
  • Reference or claim to any specific antigen herein includes all deletion, insertion and substitution mutations of that antigen, or other specific variant of that antigen as described herein, or (where the antigen is a polypeptide) to polypeptides having 80% or more, suitably at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to that polypeptide, suitably being immunogenic.
  • immunogenic composition comprising both antigens herein is intended to encompass true combinations of different antigens for combined delivery, for example in the form of a single vaccine dose, as well as an immunogenic composition comprising both antigens for simultaneous delivery, or substantially simultaneous delivery (for example by an injection of each component on the same visit to a medical practitioner), as well as a sequential delivery of one antigen followed, after a time interval, with delivery of a second antigen.
  • the immunogenic composition of the disclosure may be unitary or comprise separable components for combined or sequential delivery, as appropriate.
  • the antigen capable of generating an antibody response against a Neisseria meningitidis L2 immunotype may be any suitable antigen capable of generating an immune response which protects or ameliorates the infection or disease caused by infection by >50%, >60%, >70%, >80% or >90% of L2 immunotypes, suitably an antigen capable of generating an immune response which protects against or ameliorates the infection or disease caused by infection by the L2 immunotype.
  • the antigen is one which is encoded or expressed by >50%, >60%, >70%, >80% or >90% of Neisseria meningitidis L2 immunotypes, more suitably substantially all of Neisseria meningitidis L2 immunotypes, and more suitably wherein the % expression is determined in respect of the strains circulating in a given country or region.
  • the antigen is selected from L2 LOS, Tdfl, Hsf or Hap, or is selected from the group consisting of a combination of two or more of said antigens such as L2 LOS and Tdfl, L2 LOS and Hap, L2 LOS and Hsf, Tdfl and Hap, Tdfl and Hsf, Hap and Hsf, Tdfl and Hap and Hsf, L2 LOS and Tdfl and Hap, L2 LOS and Tdfl and Hsf, and L2 LOS and Hap and Hsf.
  • Hsf has a structure that is common to autotransporter proteins.
  • Hsf from N. meningitides strain H44/76 consists of a signal sequence made up of amino acids 1-51, a head region at the amino terminus of the mature protein (amino acids 52-479) that is surface exposed and contains variable regions (amino acids 52-106, 121-124, 191-210 and 230-234), a neck region (amino acids 480-509), a hydrophobic alpha-helix region (amino acids 518-529) and an anchoring domain in which four transmembrane strands span the outer membrane (amino acids 539-591).
  • Hsf full length Hsf may be used in immunogenic compositions of the disclosure, various Hsf truncates and deletions may also be used depending on the type of immunogenic composition or vaccine.
  • Hsf is used in a subunit composition or vaccine
  • a portion of the soluble passenger domain may be used; for instance the complete domain of amino acids 52 to 479, most suitably a conserved portion thereof, for instance the sequence of amino acids 134 to 479.
  • Suitable forms of Hsf may be truncated so as to delete variable regions of the protein disclosed in WO01/55182.
  • Suitable variants would include the deletion of one, two, three, four, or five variable regions as defined in WO01/55182.
  • the above sequences and those described below, can be extended or truncated by up to 1, 3, 5, 7, 10 or 15 amino acids at either or both N or C termini.
  • Suitable fragments of Hsf therefore include the entire head region of Hsf, suitably containing amino acids 52-473. Additional suitable fragments of Hsf include surface exposed regions of the head including one or more of the following amino acid sequences; 52-62, 76-93, 116-134, 147-157, 157-175, 199-211, 230-252, 252-270, 284-306, 328-338, 362-391, 408-418, 430-440 and 469-479.
  • Hsf is present in an outer membrane vesicle preparation, it may be expressed as the full-length protein or as a variant made up of a fusion of amino acids 1-51 and 134-591 (yielding a mature outer membrane protein of amino acid sequence 134 to the C-terminus). Suitable forms of Hsf may be truncated so as to delete variable regions of the protein disclosed in WO01/55182. Suitable variants would include the deletion of one, two, three, four, or five variable regions as defined in WO01/55182. In one aspect the first and second variable regions are deleted.
  • Suitable variants would delete residues from between amino acid sequence 52 through to 237 or 54 through to 237, more suitably deleting residues between amino acid 52 through to 133 or 55 through to 133.
  • the mature protein would lack the signal peptide.
  • Hap Computer analysis of the Hap-like protein from Neisseria meningitidis reveals at least three structural domains. Considering the Hap-like sequence from strain H44/76 as a reference, Domain 1, comprising amino-acid 1 to 42, encodes a sec-dependant signal peptide characteristic of the auto-transporter family, Domain 2, comprising amino-acids 43 to 950, encode the passenger domain likely to be surface exposed and accessible to the immune system, Domain 3, comprising residues 951 to the C-terminus (1457), is predicted to encode a beta-strands likely to assemble into a barrel-like structure and to be anchored into the outer-membrane.
  • domains 2 Since domains 2 is likely to be surface-exposed, well conserved (more than 80% in all strain tested) and could be produced as subunit antigens in E. coli , it represents an interesting vaccine candidate. Since domains 2 and 3 are likely to be surface-exposed, are well conserved (Pizza et al. (2000), Science 287: 1816-1820), they represent interesting vaccine candidates. Domain 2 is known as the passenger domain.
  • Immunogenic compositions of the disclosure may comprise the full-length Hap protein, suitably incorporated into an OMV preparation. Immunogenic compositions of the disclosure may also comprise the passenger domain of Hap which in strain H44/76 is composed of amino acid residues 43-950, or the N-terminal fragment from residues 43-1178. These fragments of Hap would be particularly advantageously used in a subunit composition of the disclosure.
  • the above sequence for the passenger domain of Hap (or N-terminal fragment) can be extended or truncated by up to 1, 3, 5, 7, 10, 15, 20, 25, or 30 amino acids at either or both N or C termini.
  • Neisserial antigen NMB0964 (NMB numbers refer to Neisseria meningitidis group B genome sequences available from www.neisseria.org) [known as NMA1161 in the Neisseria meningitidis group A genome of strain Z2491, and as BASB082 in WO 00/55327, and as ZnuD] is a conserved antigen throughout Neisseria and can induce bactericidal antibodies against a range of neisserial strains. The inventors have found this antigen functions as a Zn 2+ receptor in the bacterium, and its expression is regulated by the level of Zn 2+ in the medium.
  • NMB0964 polypeptide herein it includes the neisserial Tdfl polypeptide (encoded by the tdfl gene) in general from any neisserial strain (the protein is so well conserved amongst neisserial strains its identity in any particular neisserial strain is readily ascertainable by persons skilled in the art).
  • the term therefore includes the NMA1161 sequence, and the BASB082 polypeptide sequence (and all the Polypeptides of the Disclosure concerning the BASB082 polypeptide) of WO 00/55327.
  • NMB0964 polypeptide of the disclosure will cover SEQ ID NO: 2 of WO00/55327 or polypeptides with more than 70, 80, 90 or 95% sequence identity with said SEQ ID NO:2, or polypeptides comprising immunogenic fragments of 7, 10, 12, 15 or 20 (or more) contiguous amino acids from said SEQ ID NO: 2 (in particular said immunogenic fragments being capable of eliciting—if necessary when coupled to a protein carrier—an immune response which can recognise said SEQ ID NO: 2).
  • Particularly suitable NMB0964 immunogenic fragment embodiments are those extracellular loop sequences shown in the topology diagram of FIG. 3 as applied to any given NMB0964 sequence.
  • Said NMB0964 immunogenic fragment polypeptide sequences may have more than 70, 80, 90 or 95% sequence identity with said extracellular loop sequences (as defined in FIG. 3 ) from SEQ ID NO:2 of WO 00/55327, or may be polypeptides comprising immunogenic fragments of 7, 10, 12, 15 or 20 (or more) contiguous amino acids from said extracellular loop sequences (as defined in FIG.
  • SEQ ID NO: 2 in particular said immunogenic fragments being capable of eliciting—if necessary when coupled to a protein carrier—an immune response which can recognise said SEQ ID NO: 2) and are provided as NMB0964 polypeptides of the disclosure.
  • Said NMB0964 immunogenic fragment polypeptide sequences may have more than 70, 80, 90, 95, 99 or 100% sequence identity with the sequence from the third extracellular loop sequence given in FIG.
  • NMB0964 immunogenic fragment polypeptides are not full-length NMB0964 (mature sequence or with signal sequence) polypeptides.
  • NMB0964 may be used as an isolated antigen in a subunit composition or vaccine approach.
  • the NMB0964 antigen may be used in the form of isolated outer membrane vesicles prepared from a Neisseria species bacterium, wherein the Neisseria species bacterium produces a level of a NMB0964 polypeptide sufficient to provide for production of a vesicle that, when administered to a subject, elicits anti-NMB0964 antibodies; and a pharmaceutically acceptable excipient.
  • NMB0964 polypeptide production by for instance: disrupting the functional expression of the Zur repressor (NMB1266)—a protein which switches off expression of NMB0964 in the presence of Zn 2+ in the medium; replacing the NMB0964 promoter with one that does not bind Zur, in particular with a stronger promoter than the endogenous NMB0964 promoter such as a lac promoter; or through using a medium low in Zn 2+ concentration—i.e.
  • ⁇ M free Zn 2+ (such as Roswell Park Memorial Institute medium 1640 (RPMI) which has around 1.69 ⁇ M Zn 2+ by ICP-MS), or removing Zn 2+ in the medium, for instance using a known zinc chelator such as TPEN (N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine)—enough should be added to the medium such that the expression of the NMB0964 is maximised.
  • RPMI Roswell Park Memorial Institute medium 1640
  • TPEN N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine
  • the Neisseria species bacterium may be deficient in capsular polysaccharide, for instance through disruption of functional expression of the siaD gene. It may be disrupted in the functional expression of the msbB and/or htrB genes to detoxify the LOS in the outer membrane vesicle. It may be disrupted in the expression of one or more the following genes: PorA, PorB, OpA, OpC, PilC, or FrpB. It may be disrupted in the functional expression of the IgtB gene. Such disruption methods are described in WO 01/09350 and WO2004/014417.
  • the Neisseria species bacterium may be of immunotype L2 or L3.
  • outer membrane vesicles also known as microvesicles or blebs
  • outer membrane vesicles are well known in the art, and are described in WO 01/09350 and WO2004/014417, and also below.
  • outer membrane vesicles are isolated by extracting either without a detergent, or with 0-0.5, 0.02-0.4, 0.04-0.3, 0.06-0.2, or 0.08-0.15% detergent, for instance deoxycholate, e.g. with around or exactly 0.1% deoxycholate.
  • OMV composition or vaccine prepared either in specific culture conditions low in Zn2+, or from a mutant N. meningitidis strain engineered to either over-express NMB0964 or to remove the Zinc repression mechanism mediated through Zur, is enriched in NMB0964, and such OMVs may elicit good bactericidal antibody responses compared to OMVs which have not been prepared with these methods.
  • the disclosure relates to an immunogenic composition
  • an immunogenic composition comprising an isolated outer membrane vesicles prepared from a Neisseria species bacterium, wherein the Neisseria species bacterium produces a level of a NMB0964 polypeptide sufficient to provide for production of a vesicle that, when administered to a subject, elicits anti-NMB0964 antibodies; and a pharmaceutically acceptable excipient.
  • the NMB0964 polypeptide may be endogenous to the Neisseria species bacterium.
  • the Neisseria species bacterium may be genetically modified to contain a nucleic acid encoding an exogenous
  • NMB0964 polypeptide The NMB0964 polypeptide may be expressed from an NMB0964 gene with an endogenous promoter.
  • the Neisseria species bacterium may be genetically modified in NMB0964 polypeptide production.
  • the Neisseria species bacterium may be genetically modified through the disruption of functional expression of the Zur repressor (NMB1266).
  • the Neisseria species bacterium may be genetically modified to provide for expression of a NMB0964 polypeptide from a heterologous promoter.
  • the heterologous promoter in one aspect does not bind the Zur repressor.
  • the heterologous promoter is a stronger promoter in the Neisserial species bacterium than the non-repressed endogenous promoter of the NMB0964 gene.
  • the heterologous promoter is an IPTG-inducible lac promoter.
  • the level of NMB0964 polypeptide produced by the Neisseria species bacterium is greater than that made by N. meningitidis strain H44/76 grown in tryptic soy broth (TSB). In one aspect level of NMB0964 polypeptide produced by the Neisseria species bacterium is the same or greater than that made by N. meningitidis strain H44/76 grown in Roswell Park Memorial Institute medium 1640 (RPMI). In one aspect the level of NMB0964 polypeptide produced by the Neisseria species bacterium is the same or greater than that made by N.
  • NMB0964 polypeptide produced by the Neisseria species bacterium is the same or greater than that made by N. meningitidis strain H44/76 in a medium which has less than 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05 or 0.01 ⁇ M free Zn 2+ .
  • the Neisserial species bacterium is Neisseria meningitidis , or Neisseria meningitidis serogroup B. In one aspect the Neisseria species bacterium is deficient in capsular polysaccharide. In one aspect the Neisseria species bacterium is deficient in capsular polysaccharide through disruption of functional expression of the siaD gene. In one aspect the Neisseria species bacterium is disrupted in the functional expression of the msbB and/or htrB genes. In one aspect the Neisseria species bacterium is disrupted in the expression of one or more the following genes: PorA, PorB, OpA, OpC, PilC, or FrpB. In one aspect the Neisseria species bacterium is disrupted in the functional expression of the IgtB gene. In one aspect wherein the Neisseria species bacterium is of immunotype L2 or L3.
  • the outer membrane vesicles are isolated by extracting with 0-0.5, 0.02-0.4, 0.04-0.3, 0.06-0.2, or 0.08-0.15% detergent, for instance deoxycholate, e.g. with around or exactly 0.1% deoxycholate.
  • the disclosure relates to a method of producing an immunogenic composition, the method comprising: culturing a Neisseria species bacterium producing a NMB0964 polypeptide, wherein the NMB0964 polypeptide is produced at a level sufficient to provide for production of outer membrane vesicles that, when administered to a subject, elicit anti-NMB0964 antibodies; preparing outer membrane vesicles from the cultured bacterium; and combining the outer membrane vesicles with a pharmaceutically acceptable excipient to produce an immunogenic composition suitable for administration to a subject in combination with an fHbp polypeptide.
  • the NMB0964 polypeptide is endogenous to the Neisseria species bacterium.
  • the Neisseria species bacterium has been genetically modified to contain a nucleic acid encoding an exogenous NMB0964 polypeptide.
  • the NMB0964 polypeptide is expressed from an NMB0964 gene with an endogenous promoter.
  • the Neisseria species bacterium has been genetically modified in NMB0964 polypeptide production.
  • the Neisserial species bacterium has been genetically modified through the disruption of functional expression of the Zur repressor (NMB1266).
  • the Neisseria species bacterium has been genetically modified to provide for expression of a NMB0964 polypeptide from a heterologous promoter.
  • heterologous promoter does not bind the Zur repressor.
  • heterologous promoter is a stronger promoter in the Neisserial species bacterium than the non-repressed endogenous promoter of the NMB0964 gene.
  • the heterologous promoter is an IPTG-inducible lac promoter.
  • the level of NMB0964 polypeptide produced by the Neisseria species bacterium is greater than that made by N. meningitidis strain H44/76 grown in tryptic soy broth (TSB).
  • TTB tryptic soy broth
  • the level of NMB0964 polypeptide produced by the Neisseria species bacterium is the same or greater than that made by N.
  • the level of NMB0964 polypeptide produced by the Neisseria species bacterium is the same or greater than that made by N. meningitidis strain H44/76 grown in Roswell Park Memorial Institute medium 1640 (RPMI) with 1 ⁇ M TPEN (N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine). In one aspect the level of NMB0964 polypeptide produced by the Neisseria species bacterium is the same or greater than that made by N.
  • meningitidis strain H44/76 in a medium which has less than 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05 or 0.01 ⁇ M free Zn 2+ .
  • culturing of the Neisseria species bacterium is in a medium which has less than 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05 or 0.01 ⁇ M free Zn 2+ .
  • culturing of the Neisserial species bacterium is in a medium comprising a Zn 2+ chelator.
  • Zn 2+ chelator is present in the medium at a concentration of 0.01-100, 0.1-10, 0.3-5, or 0.5-1 ⁇ M.
  • the Zn 2+ chelator present in the medium is TPEN, suitably at a concentration in the range of 1 to 25 ⁇ M, suitably a concentration of 5 ⁇ M, 10 ⁇ M, 15 ⁇ M, 20 ⁇ M or 25 ⁇ M.
  • the step of preparing outer membrane vesicles is carried out by extracting with 0-0.5, 0.02-0.4, 0.04-0.3, 0.06-0.2, or 0.08-0.15% detergent, for instance deoxycholate, e.g. with around or exactly 0.1-0.5% deoxycholate.
  • the step of preparing outer membrane vesicles is carried out without use of a detergent.
  • the fHbp polypeptide is combined with a peptide sequence sharing more than 50, 60, 70, 80, 90, 95, 99, or of 100% sequence identity with the following sequence: RDQYGLPAHSHEYDDCHADIIWQKSLINKRYLQLYPHLLTEEDIDYDNPGLSCGFHDDDNA HAHTHS, or a polypeptide comprising an immunogenic fragment of 7, 10, 12, 15 or 20 (or more) contiguous amino acids from said sequence (optionally wherein said peptide sequence or said immunogenic fragment is capable of eliciting—if necessary when coupled to a protein carrier—an immune response which can recognise SEQ ID NO: 2 of WO00/55327), and a pharmaceutically acceptable carrier.
  • the two Cys residues are present in the polypeptide, and may di-sulphide linked.
  • the polypeptide is not a full length mature NMB0964 polypeptide, or is not a full length NMB0964 polypeptide with signal sequence intact.
  • L2 Lipooligosaccharide L2 LOS
  • LPS lipopolysaccharide, also known as LOS-lipooligosaccharide
  • LPS lipopolysaccharide, also known as LOS-lipooligosaccharide
  • the polysaccharide moiety of the LPS is known to induce bactericidal antibodies.
  • the structure and function of Los is described inn Verheul Microbiological reviews, March 1993, Vol 57, no 1, p34-49.
  • Meningococcal LPS L3, 7, 9 (L3), L2 and L5 can be modified by sialylation, or by the addition of cytidine 5′-monophosphate-N-acetylneuraminic acid. See M. P. Jennings et al, Microbiology 1999, 145, 3013-3021 and Mol Microbiol 2002, 43: 931-43 for further illustration of LPS structure and heterogeneity.
  • L2 LOS defines the L2 immunotype and thus is a potential antigen to supplement an fHbp based composition or vaccine.
  • L2 LOS suitably generates antibodies capable of killing a Neisserial L2 immunotype.
  • L2 LOS herein includes L3V strains.
  • L2 LOS may be presented in an outer membrane vesicle (OMV—the term ‘bleb’ and ‘outer membrane vesicle’ are used interchangeably herein), suitably where the vesicle is extracted with a low percentage detergent, more suitably 0-0.5%, 0.02-0.4%, 0.04-0.3%, 0.06-0.2%, 0.08-0.15% or 0.1%, most suitably deoxycholate [DOC]) but may also be part of a subunit composition or vaccine.
  • OMV outer membrane vesicle
  • OMVs of the present disclosure may be extracted using a low percentage detergent, more suitably 0-0.5%, 0.02-0.4%, 0.04-0.3%, 0.06-0.2%, 0.08-0.15% or 0.1%, most suitably deoxycholate [DOC])
  • LOS may be used plain or conjugated to a source of T-cell epitopes such as tetanus toxoid, Diphtheria toxoid, CRM-197 or OMV outer membrane proteins.
  • L2 LOS may be detoxified.
  • LPS suitably meningococcal LPS
  • a composition or vaccine of the disclosure suitably either or both of immunotypes L2 and L3 are present.
  • the second antigen is a meningococcal LPS having a least a PEA on position 6 on Hep II of inner core with or without a second PEA or a glucose on this Hep II.
  • the combination of the disclosure may be used with other antigens for combined, simultaneous or sequential delivery.
  • the combination of the disclosure includes an antigen that is also effective against ST269, which reference includes the ST269 clonal complex.
  • the antigen effective against ST269 clonal complex is selected from Hap, Tdfl and Hsf (discussed in more detail below), or is selected from the group consisting of a combination of two or more of said antigens such as Tdfl and Hap, Tdfl and Hsf, Hap and Hsf, and Tdfl and Hap and Hsf.
  • the combination of the disclosure includes an antigen that is also effective against ST11, which reference includes the ST11 clonal complex.
  • the antigen effective against ST11 clonal complex is selected from L2 LOS, Hap, Tdfl and Hsf (discussed in more detail below), or is selected from the group consisting of a combination of two or more of said antigens such as L2 LOS and Tdfl, L2 LOS and Hap, L2 LOS and Hsf, Tdfl and Hap, Tdfl and Hsf, Hap and Hsf, Tdfl and Hap and Hsf, L2 LOS and Tdfl and Hap, L2 LOS and Tdfl and Hap, L2 LOS and Tdfl and Hsf, and L2 LOS and Hap and Hsf.
  • Sequence types are identified by Multilocus sequence typing (MLST), which characterises isolates of bacterial species using the sequences of internal fragments of seven house-keeping genes. For each house-keeping gene, the different sequences present within a bacterial species are assigned as distinct alleles and, for each isolate, the alleles at each of the seven loci define the allelic profile or sequence type (ST). Sequence types are grouped into clonal complexes by their similarity to a central allelic profile (genotype). For Neisseria , generally once a central genotype has been identified, clonal complexes are defined as including any ST that matches the central genotype at four or more loci unless it more closely matches another central genotype. Reference to Neisserial meningitidis ST269 or ST11 herein includes reference to Neisserial meningitidis ST269 or ST11 clonal complexes, respectively.
  • MMT Multilocus sequence typing
  • suitable immunogenic compositions of the disclosure are capable of protecting against infection or disease caused by >50%, suitably >60%, suitably >70%, suitably >80%, suitably >90% of Neisseria meningitidis B strains.
  • suitable immunogenic compositions of the disclosure are capable of protecting against infection or disease caused by >50%, suitably >60%, suitably >70%, suitably >80%, suitably >90% of the following group of Neisseria meningitidis B clonal complexes: ST41/44, ST269, ST213.
  • the antigen capable of generating an antibody response against a Neisseria meningitidis ST269 strain may be any suitable antigen, suitably an antigen capable of generating an immune response which protects or ameliorates the infection or disease caused by infection with ST269.
  • an antigen which is effective against ST269 is an antigen capable of protecting against infection or disease caused by >50%, >60%, >70%, >80%, >90% of Neisseria meningitidis ST269, more suitably substantially all of Neisseria meningitidis ST269 clonal types.
  • the antigen capable of generating an antibody response against a Neisseria meningitidis ST 11 strain may be any suitable antigen, suitably an antigen capable of generating an immune response which protects or ameliorates the infection or disease caused by infection with ST11.
  • an antigen which is effective against ST11 is an antigen capable of protecting against infection or disease caused by >50%, >60%, >70%, >80%, >90% of Neisseria meningitidis ST11, more suitably substantially all of Neisseria meningitidis ST11 clonal types.
  • Suitable other antigens may be selected from categories of proteins including adhesins, autotransporter proteins, toxins, integral outer membrane proteins and Fe or Zn acquisition proteins.
  • the disclosure provides immunogenic compositions that comprise at least or exactly two, three, four, five, six, seven, eight, nine or ten different Neisseria antigens. Most suitably these antigens are selected from at least or exactly two, three, four or five groups of proteins selected from the following:
  • a Neisserial adhesin selected from the group consisting of FhaB, NspA PilC, Hsf, Hap, MafA, MafB, Omp26, NMB 0315, NMB 0995, NMB 1119 and NadA
  • a Neisserial autotransporter selected from the group consisting of Hsf, Hap, IgA protease, AspA, and NadA
  • a Neisserial toxin selected from the group consisting of FrpA, FrpC, FrpA/C, VapD, NM-ADPRT and either or both of LPS immunotype L2 and LPS immunotype L3
  • a Neisserial Fe or zinc acquisition protein or other metal acquisition protein selected from the group consisting of TbpA, TbpB, LbpA, LbpB, HpuA, HpuB, Lipo28 (GNA2132), Sibp, NMB0964, NMB0293, FbpA, Bcp, BfrA, Bf
  • the antigens of the present disclosure are all isolated, meaning that they are altered by the hand of man. In one aspect they are purified to some degree, most suitably more than 40, 50, 60, 70, 80, 90, 95 or 99% pure (before combination with the other components of the immunogenic compositions of the disclosure).
  • a protein is specifically mentioned herein, it is suitably a reference to a native, full-length protein, and to its natural variants (i.e. to a native protein obtainable from a Neisserial, suitably meningococcal strain) but it may also encompass antigenic fragments thereof (particularly in the context of subunit vaccines). These are fragments (often specifically described herein) containing or comprising at least 15 amino acids, suitably at least 20 amino acids, at least 30 amino acids, at least 40 amino acids or at least 50 amino acids, taken contiguously from the amino acid sequence of the protein. Antigenic fragments may also be immunogenic fragments.
  • reference to proteins and protein sequences herein includes a polypeptide comprising an immunogenic fragment of 7, 10, 12, 15, 20, 30, 40 or 50 (or more) contiguous amino acids from said protein sequence or from the amino acid sequence of said protein (optionally wherein said immunogenic fragment is capable of eliciting—if necessary when coupled to a protein carrier—an immune response which can recognise said protein or said protein sequence).
  • antigenic fragments denotes fragments that are immunologically reactive with antibodies generated against the Neisserial full-length proteins or with antibodies generated by infection of a mammalian host with Neisseria .
  • Antigenic fragments also includes fragments that when administered at an effective dose, elicit a protective immune response against Neisserial infection, more suitably it is protective against N. meningitidis and/or N. gonorrhoeae infection, most suitably it is protective against N. meningitidis serogroup B infection.
  • recombinant fusion proteins of Neisserial proteins are also included. These may combine different Neisserial proteins or fragments thereof in the same polypeptide.
  • the disclosure also includes individual fusion proteins of Neisserial proteins or fragments thereof, as a fusion protein with heterologous sequences such as a provider of T-cell epitopes or purification tags, for example: p-galactosidase, glutathione-S-transferase, green fluorescent proteins (GFP), epitope tags such as FLAG, myc tag, poly histidine, or viral surface proteins such as influenza virus haemagglutinin, tetanus toxoid, diphtheria toxoid, CRM197.
  • heterologous sequences such as a provider of T-cell epitopes or purification tags, for example: p-galactosidase, glutathione-S-transferase, green fluorescent proteins (GFP), epitope tags such as FLAG, myc tag, poly hist
  • Antigens of the disclosure associated with NMB references refer to known reference numbers corresponding to known sequences (well-known to a skilled person) which can (for example) be accessed from www.neisseria.org.
  • Adhesins include FhaB (WO98/02547), NadA (J. Exp. Med (2002) 195: 1445; NMB 1994), Hsf also known as NhhA (NMB 0992) (WO99/31132), Hap (NMB 1985) (WO99/55873), NspA (WO96/29412), MafA (NMB 0652) and MafB (NMB 0643) (Annu Rev Cell Dev Biol. 16; 423-457 (2000); Nature Biotech 20; 914-921 (2002)), Omp26 (NMB 0181), NMB 0315, NMB 0995, NMB 1119 and PilC (Mol. Microbiol. 1997, 23; 879-892).
  • Hsf is an example of an adhesin, as well as being an autotranporter protein.
  • Immunogenic compositions of the disclosure may therefore include combinations of Hsf and other autotransporter proteins where Hsf contributes in its capacity as an adhesin.
  • adhesins may be derived from Neisseria meningitidis or Neisseria gonorrhoeae or other Neisserial strains.
  • the disclosure also includes other adhesins from Neisseria.
  • FhaB This antigen has been described in WO98/02547 SEQ ID NO 38 (nucleotides 3083-9025)—see also NMB0497.
  • the present inventors have found FhaB to be particularly effectively at inducing anti-adhesive antibodies alone and in particular with other antigens of the disclosure.
  • full length FhaB could be used, the inventors have found that particular C-terminal truncates are surprisingly at least as effective and suitably even more effective in terms of cross-strain effect. Such truncates have also been advantageously shown to be far easier to clone.
  • FhaB truncates of the disclosure typically correspond to the N-terminal two-thirds of the FhaB molecule, suitably the new C-terminus being situated at position 1200-1600, more suitably at position 1300-1500, and most suitably at position 1430-1440. Specific embodiments have the C-terminus at 1433 or 1436. Accordingly such FhaB truncates of the disclosure and vaccines comprising such truncates are independent aspects of the present disclosure as well as being components of the combination immunogenic compositions of the disclosure.
  • the N-terminus may also be truncated by up to 10, 20, 30, 40 or 50 amino acids.
  • Autotransporter proteins typically are made up of a signal sequence, a passenger domain and an anchoring domain for attachment to the outer membrane.
  • autotransporter proteins include Hsf (WO99/31132) (NMB 0992), HMW, Hia (van Ulsen et al Immunol. Med. Microbiol. 2001 32; 53-64), Hap (NMB 1985) (WO99/55873; van Ulsen et al Immunol. Med. Microbiol. 2001 32; 53-64), UspA, UspA2, NadA (NMB 1994) (Comanducci et al J. Exp. Med.
  • NadA J. Exp. Med (2002) 195: 1445
  • Immunogenic compositions of the disclosure may therefore include combinations of NadA and adhesins where NadA contributes in its capacity as an autotransporter protein.
  • These proteins may be derived from Neisseria meningitidis or Neisseria gonorrhoeae or other Neisserial strains.
  • the disclosure also includes other autotransporter proteins from Neisseria.
  • Metal Acquisition Proteins Such as Iron and Zinc Acquisition Proteins:
  • Tdfl NMB0964
  • TdfH NmB1497
  • TbpA NMB 0461
  • TbpB NMB 0460
  • LbpA NMB 1540
  • LbpB NMB 1541
  • HpuA U73112.2
  • TbpA interacts with TbpB to form a protein complex on the outer membrane of Neisseria , which binds transferrin.
  • TbpA contains an intracellular N-terminal domain with a TonB box and plug domain, multiple transmembrane beta strands linked by short intracellular and longer extracellular loops.
  • TbpB Two families of TbpB have been distinguished, having a high molecular weight and a low molecular weight respectively.
  • High and low molecular weight forms of TbpB associate with different families of TbpA which are distinguishable on the basis of homology.
  • they are known as the high molecular weight and low molecular weight families because of their association with the high or low molecular weight form of TbpB (Rokbi et al FEMS Microbiol. Lett.
  • humanogenic compositions of the disclosure may comprise TbpA and TbpB from serogroups A, B, C, Y and W-135 of N. meningitidis as well as iron acquisition proteins from other bacteria including N. gono7rhoeae. Transferrin binding proteins TbpA and TbpB have also been referred to as Tbp1 and Tbp2 respectively (Cornelissen et al Infection and Immunity 65; 822, 1997).
  • TbpA contains several distinct regions.
  • the amino terminal 186 amino acids form an internal globular domain, 22 beta strands span the membrane, forming a beta barrel structure.
  • Extracellular loops 2, 3 and 5 have the highest degree of sequence variability and loop 5 is surface exposed. Loops 5 and 4 are involved in ligand binding.
  • Suitable fragments of TbpA include the extracellular loops of TbpA. Using the sequence of TbpA from N. meningitidis strain H44/76, these loops correspond to amino acids 200-202 for loop 1, amino acids 226-303 for loop 2, amino acids 348-395 for loop 3, amino acids 438-471 for loop 4, amino acids 512-576 for loop 5, amino acids 609-625 for loop 6, amino acids 661-671 for loop 7, amino acids 707-723 for loop 8, amino acids 769-790 for loop 9, amino acids 814-844 for loop 10 and amino acids 872-903 for loop 11.
  • the corresponding sequences, after sequence alignment, in other Tbp proteins would also constitute suitable fragments. Most suitable fragments would include amino acid sequences constituting loop 2, loop 3, loop 4 or loop 5 of Tbp.
  • immunogenic compositions of the disclosure comprise TbpA
  • TbpA is suitably presented in an outer membrane vesicle (OMV) vaccine, it may also be part of a subunit vaccine.
  • OMV outer membrane vesicle
  • isolated iron acquisition proteins which could be introduced into an immunogenic composition of the disclosure are well known in the art (WO00/25811). They may be expressed in a bacterial host, extracted using detergent (for instance 2% Elugent) and purified by affinity chromatography or using standard column chromatography techniques well known to the art (Oakhill et al Biochem J. 2002 364; 613-6).
  • TbpA is presented in an OMV vaccine
  • its expression can be upregulated by genetic techniques discussed herein, or may suitably be upregulated by growth of the parent strain under iron limitation conditions as described below.
  • This process will also result in the upregulation of variable iron-regulated proteins, particularly FrpB which may become immunodominant and it is therefore advantageous to downregulate the expression of (and suitably delete the genes encoding) such proteins (particularly FrpB) as described below, to ensure that the immunogenic composition of the disclosure elicits an immune response against antigens present in a wide range of Neisserial strains.
  • Toxins include FrpA (NMB 0585; NMB 1405), FrpA/C (see below for definition), FrpC (NMB 1415; NMB 1405) (WO92/01460), NM-ADPRT (NMB 1343) (13′ h International Pathogenic Neisseria Conference 2002 Masignani et al p135), VapD (NMB 1753), lipopolysaccharide (LPS; also called lipooligosaccharide or LOS) immunotype L2 and LPS immunotype L3.
  • FrpA NMB 0585; NMB 1405
  • FrpA/C see below for definition
  • FrpC NMB 1415; NMB 1405)
  • NM-ADPRT NMB 1343
  • LPS lipopolysaccharide
  • LPS lipooligosaccharide
  • FrpA and FrpC contain a region which is conserved between these two proteins and a suitable fragment of the proteins would be a polypeptide containing this conserved fragment, suitably comprising amino acids 227-1004 of the sequence of FrpA/C.
  • These antigens may be derived from Neisseria fyzeningitidis or Neisseria gonorrlaoeae or other Neisserial strains.
  • the disclosure also includes other toxins from Neisseria.
  • toxins may include antigens involved in the regulation of toxicity, for example OstA which functions in the synthesis of lipopolysaccharides.
  • FrpA and FrpC Neisseria 7nenngtds encodes two RTX proteins, referred to as FrpA & FrpC secreted upon iron limitation (Thompson et al., (1993) J. Bacteriol. 175: 811-818; Thompson et al., (1993) Infect. Immun. 61: 2906-2911).
  • the RTX (Repeat ToXin) protein family have in common a series of 9 amino acid repeat near their C-termini with the consensus: Leu Xaa Gly Gly Xaa Gly (Asn/Asp) Asp Xaa (LXGGXGN/DDX). The repeats in E.
  • coli HlyA are thought to be the site of Ca2+ binding.
  • Immunogenic compositions of the disclosure may comprise the full length FrpA and/or FrpC or suitably, a fragment comprising the sequence conserved between FrpA and FrpC.
  • the conserved sequence is made up of repeat units of 9 amino acids.
  • Immunogenic compositions of the disclosure would suitably comprise more that three repeats, more than 10 repeats, more than 13 repeats, more than 20 repeats or more than 23 repeats.
  • Such truncates have advantageous properties over the full length molecules and composition or vaccines comprising such antigens form an independent aspect of disclosure as sell as being incorporated in the immunogenic compositions of the disclosure.
  • FrpA/C Sequences conserved between FrpA and FrpC are designated FrpA/C and wherever FrpA or FrpC forms a constituent of immunogenic compositions of the disclosure, FrpA/C could be advantageously used.
  • Amino acids 277-1004 of the FrpA sequence is the suitable conserved region. The above sequence can be extended or truncated by up to 1, 3, 5, 7, 10, 15, 20, 25, or 30 amino acids at either or both N or C termini.
  • L2 LOS is combined with fHbp.
  • LPS is suitably presented in an outer membrane vesicle (OMV) (suitably where the vesicle is extracted with a low percentage detergent, more suitably 0-0.5%, 0.02-0.4%, 0.04-0. 3%, 0.06-0.2%, 0.08-0.15% or 0.1%, most suitably deoxycholate [DOC]) but may also be part of a subunit vaccine.
  • OMV outer membrane vesicle
  • LPS may be isolated using well known precedure including the hot water-phenol procedure (Wesphal and Jann Meth. Carbo. Chem. 5; 83-91 1965). See also Galanos et al. 1969, Eur J Biochem 9: 245-249, and Wu et al. 1987, Anal Bio Chem 160: 281-289.
  • LPS may be used plain or conjugated to a source of T-cell epitopes such as tetanus toxoid, Diphtheria toxoid, CRM-197 or OMV outer membrane proteins.
  • T-cell epitopes such as tetanus toxoid, Diphtheria toxoid, CRM-197 or OMV outer membrane proteins.
  • Techniques for conjugating isolated LOS are also known (see for instance EP 941738 incorporated by reference herein).
  • LOS in particular the LOS of the disclosure
  • the LOS is suitably conjugated in situ by methods allowing the conjugation of LOS to one or more outer membrane proteins also present on the bleb preparation (e.g. PorA or PorB in meningococcus).
  • This process can advantageously enhance the stability and/or immunogenicity (providing T-cell help) and/or antigenicity of the LOS antigen within the bleb formulation-thus giving T-cell help for the T-independent oligosaccharide immunogen in its most protective conformation-as LOS in its natural environment on the surface of meningococcal outer membrane.
  • conjugation of the LOS within the bleb can result in a detoxification of the LOS (the Lipid A portion being stably buried in the outer membrane thus being less available to cause toxcity).
  • compositions comprising blebs wherein LOS present in the blebs has been conjugated in an intra-bleb fashion to outer membrane proteins also present in the bleb can form the basis of a composition or vaccine for the treatment or prevention of diseases caused by the organism from which the blebs have been derived, wherein such vaccine is substantially non-toxic and is capable of inducing a T-dependent bactericidal response against LOS in its native environment.
  • This disclosure therefore further provides such an intra-bleb LOS conjugated meningococcal bleb preparation.
  • Such bleb preparations may be isolated from the bacterial in question (see WO 01/09350), and then subjected to known conjugation chemistries to link groups (e.g. NH2 or COOH) on the oligosaccharide portion of LOS to groups (e.g. NH2 or COOH) on bleb outer membrane proteins.
  • link groups e.g. NH2 or COOH
  • cross-linking techniques using glutaraldehyde, formaldehyde, or glutaraldehyde/formaldehyde mixes may be used, but it is suitable that more selective chemistries are used such as EDAC or EDAC/NHS (J. V. Staros, R. W. Wright and D. M. Swingle.
  • the bleb preparations are conjugated in the absence of capsular polysaccharide.
  • the blebs may be isolated from a strain which does not produce capsular polysaccharide (naturally or via mutation as described below), or may be purified from most and suitably all contaminating capsular polysaccharide. In this way, the intra-bleb LOS conjugation reaction is much more efficient.
  • more than 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% of the LOS present in the blebs is cross-linked/conjugated.
  • Intrableb conjugation should suitably incorporate 1, 2 or all 3 of the following process steps: conjugation pH should be greater than pH 7.0, suitably greater than or equal to pH 7.5 (most suitably under pH 9); conditions of 1-5% suitably 2-4% most suitably around 3% sucrose should be maintained during the reaction; NaCl should be minimised in the conjugation reaction, suitably under 0.1M, 0.05M, 0.01M, 0.005M, 0.001M, and most suitably not present at all. All these process features make sure that the blebs remain stable and in solution throughout the conjugation process.
  • the EDAC/NHS conjugation process is a suitable process for intra-bleb conjugation.
  • EDAC/NHS is suitable to formaldehyde which can cross-link to too high an extent thus adversely affecting filterability.
  • EDAC reacts with carboxylic acids (such as KDO in LOS) to create an active-ester intermediate.
  • carboxylic acids such as KDO in LOS
  • an amine nucleophile such as lysines in outer membrane proteins such as PorB
  • an amide bond is formed with release of an isourea by-product.
  • the efficiency of an EDAC-mediated reaction may be increased through the formation of a Sulfo-NHS ester intermediate.
  • the Sulfo-NEIS ester survives in aqueous solution longer than the active ester formed from the reaction of EDAC alone with a carboxylate.
  • higher yields of amide bond formation may be realized using this two-stage process.
  • EDAC/NHS conjugation is discussed in J. V. Staros, R. W. Wright and D. M. Swingle, Enhancement by N-hydroxysuccinimide of water-soluble carbodiimide-mediated coupling reactions. Analytical chemistry 156: 220-222 (1986); and Bioconjugates Techniques. Greg T. Hermanson (1996) pp 173-176.
  • 0.01-5 mg EDAC/mg bleb is used in the reaction, more suitably 0.05-1 mg EDAC/mg bleb.
  • the amount of EDAC used depends on the amount of LOS present in the sample which in turn depends on the deoxycholate (DOC) % used to extract the blebs. At low % DOC (e.g.
  • a suitable process of the disclosure is therefore a process for producing intra-bleb conjugated LOS (suitably meningococcal) comprising the steps of conjugating blebs in the presence of EDAC/NHS at a pH between pH 7.0 and pH 9.0 (suitably around pH 7.5), in 1-5% (suitably around 3%) sucrose, and optionally in conditions substantially devoid of NaCl (as described above), and isolating the conjugated blebs from the reaction mix.
  • the reaction may be followed on Western separation gels of the reaction mixture using anti-LOS (e.g. anti-L2 or anti-L3) mAbs to show the increase of LOS molecular weight for a greater proportion of the LOS in the blebs as reaction time goes on.
  • anti-LOS e.g. anti-L2 or anti-L3
  • EDAC was found to be an excellent intra-bleb cross-linking agent in that it cross-linked LOS to OMP sufficiently for improved LOS T-dependent immunogenicity, but did not cross link it to such a high degree that problems such as poor filterability, aggregation and inter-bleb cross-linking occurred.
  • the morphology of the blebs generated is similar to that of unconjugated blebs (by electron microscope).
  • the above protocol avoided an overly high cross-linking to take place (which can decrease the immunogenicity of protective OMPs naturally present on the surface of the bleb e.g. TbpA or Hsf).
  • the meningococcal strain from which the blebs are derived is a mutant strain that cannot produce capsular polysaccharide (e.g. one of the mutant strains described below, in particular siaD′).
  • immunogenic compositions effective against meningococcal disease comprise both an L2 and L3 bleb, wherein the L2 and L3 LOS are both conjugated to bleb outer membrane proteins.
  • the LOS structure within the intra-bleb conjugated bleb is consistent with it having been derived from an IgtB-meningococcal strain (as described below).
  • immunogenic compositions comprise intrableb-conjugated blebs: derived from a mutant meningococcal strain that cannot produce capsular polysaccharide and is IgtB-; comprising L2 and L3 blebs derived from mutant meningococcal strains that cannot produce capsular polysaccharide; comprising L2 and L3 blebs derived from mutant meningococcal strains that are IgtB-; or most suitably comprising L2 and L3 blebs derived from mutant meningococcal strains that cannot produce capsular polysaccharide and are IgtB-.
  • Typical L3 meningococcal strain that can be used for the present disclosure is H44/76 menB strain.
  • a typical L2 strain is the B16B6 menB strain or the 39E meningococcus type C strain.
  • the blebs of the disclosure have been detoxified to a degree by the act of conjugation, and need not be detoxified any further, however further detoxification methods may be used for additional security, for instance using blebs derived from a meningococcal strain that is htrB′ or msbB- or adding a non-toxic peptide functional equivalent of polymyxin B [a molecule with high affinity to Lipid A] (suitably SEAP 2) to the bleb composition (as described above).
  • meningococcal blebs and immunogenic compositions comprising blebs which have as an important antigen LOS which is substantially non-toxic, devoid of autoimmunity problems, has a T-dependent character, is present in its natural environment, and is capable of inducing a bactericidal antibody response against more than 90% of meningococcal strains (in the case of L2+L3 compositions).
  • intrableb LOS conjugation should incorporate 1, 2 or all 3 of the following process steps: conjugation pH should be greater than pH 7.0, suitably greater than or equal to pH 7.5 (most suitably under pH 9); conditions of 1-5% suitably 2-4% most suitably around 3% sucrose should be maintained during the reaction; NaCl should be minimised in the conjugation reaction, suitably under 0.1M, 0.05M, 0.01M, 0.005M, 0.001M, and most suitably not present at all. All these process features make sure that the blebs remain stable and in solution throughout the conjugation process.
  • EDAC/NHS conjugation process is a suitable process for intra-bleb conjugation.
  • EDAC/NHS is more suitable than formaldehyde which can cross-link to too high an extent thus adversely affecting filterability.
  • EDAC reacts with carboxylic acids to create an active-ester intermediate.
  • Neisserial proteins may also be candidates for inclusion in the Neisserial composition or vaccines of the disclosure and may be able to combine with other antigens in a surprisingly effective manner.
  • Membrane associated proteins particularly integral membrane proteins and most advantageously outer membrane proteins, especially integral outer membrane proteins may be used in the compositions of the present disclosure.
  • An example of such a protein is PIDA also known as Omp IA (NMB 0464) (WO00/15801) which is a Neisserial phospholipase outer membrane protein.
  • TspA NMB 0341
  • TspB T-cell stimulating protein
  • PilQ NMB 1812
  • OMP85 also known as D15-(NMB 0182) (WO00/23593)
  • NspA U52066
  • FhaC FhaC(NMB 0496 or NMB 1780
  • PorB NMB 2039
  • HpuB NC — 003116.1
  • TdfH NMB 1497
  • OstA NMB 0280
  • MItA also known as GNA33 and Lipo30
  • HtrA NMB 0532; WO 99/55872
  • HimD NMB 1302
  • H isD NMB 1581
  • HIpA NMB 1946
  • NMB 1124 NMB 1162, NMB 1220, NMB 1313, NMB 1953, HtrA, TbpA (NMB 0461) (WO92/03467)
  • TdfH NmB1497)
  • Tdfl NMB0964
  • OMP85 Immunogenic compositions of the disclosure may comprise the full length OMP85, suitably as part of an OMV preparation. Fragments of OMP85 may also be used in immunogenic compositions of the disclosure, in particularly, the surface exposed domain of OMP85 made up of amino acid residues 1-475 or 50-475 is suitably incorporated into a subunit component of the immunogenic compositions of the disclosure.
  • the above sequence for the surface exposed domain of OMP85 can be extended or truncated by up to 1, 3, 5, 7, 10, 15, 20, 25, or 30 amino acids at either or both N or C termini.
  • the signal sequence may be omitted from the OMP85 fragment.
  • OstA OstA functions in the synthesis of lipopolysaccharides and may be considered to be a regulator of toxicity. OstA may alternatively be included in the toxin category where the toxin category is broadened to contain regulators of toxicity as well as toxins.
  • the disclosure relates to a polynucleotide encoding a protein or combination of proteins present in the composition as claimed.
  • the fHbp may be provided in combination with a second antigen, either as a polynucleotide encoding a fusion protein or as a polynucleotide encoding polypeptides on the same nucleic acid construct (e.g. vector construct)
  • Polynucleotide generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. “Polynucleotides” include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
  • Modified bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications has been made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.
  • Polynucleotide also embraces relatively short polynucleotides, often referred to as oligonucleotides.
  • Another aspect of the disclosure relates to an immunological/vaccine formulation which comprises one or more polynucleotide(s).
  • immunological/vaccine formulation which comprises one or more polynucleotide(s).
  • composition or vaccines comprise one or more polynucleotide (s) encoding a plurality of proteins corresponding to protein combinations of the disclosure described above.
  • polynucleotides may be under the control of a eukaryotic promoter capable of driving expression within a mammalian cell.
  • the polynucleotide may additionally comprise sequence encoding other antigens.
  • eukaryotic promoters examples include viral promoters from viruses including adenoviral promoters, retroviral promoters.
  • the disclosure relates to a method for manufacture of an improved fHbp based composition or vaccine for prevention or amelioration of Neisseria meningitidis infection or disease, the method comprising combining fHbp with a second antigen capable of generating an antibody response against a Neisseria meningitidis L2 immunotype
  • composition of the disclosure may be a subunit composition, a composition comprising antigens in the context of an outer membrane vesicle (bleb), or comprise a combination of subunit and outer membrane vesicle.
  • bleb outer membrane vesicle
  • the disclosure relates to culturing a Neisseria species bacterium producing a NMB0964 polypeptide, wherein the NMB0964 polypeptide is produced at a level sufficient to provide for production of outer membrane vesicles that, when administered to a subject, elicit anti-NMB0964 antibodies; preparing outer membrane vesicles from the cultured bacterium; and combining the outer membrane vesicles with a pharmaceutically acceptable excipient to produce an immunogenic composition suitable for administration to a subject in combination with an fHbp polypeptide as defined herein.
  • the immunogenic composition or vaccine of the disclosure may be a subunit composition.
  • Subunit compositions are compositions in which the component or components have been isolated and purified to at least 50%, suitably at least 60%, 70%, 80%, 90% purity.
  • Subunit compositions may be in aqueous solution. They may comprise detergent, suitably non-ionic, zwitterionic or ionic detergent in order to solubilise hydrophobic portions of the antigens. They may comprise lipids so that liposome structures could be formed, allowing presentation of antigens with a structure that spans a lipid membrane.
  • meningitidis serogroup B excretes outer membrane blebs in sufficient quantities to allow their manufacture on an industrial scale.
  • An outer membrane vesicle may also be prepared via the process of detergent extraction of the bacterial cells (see for example EP 11243).
  • the immunogenic composition of the disclosure may also comprise an outer membrane vesicle preparation suitably having an antigen which has been been upregulated, either recombinantly or by other means including growth under iron-depleted conditions and/or under zinc depleted conditions.
  • antigens which would be upregulated in such a outer membrane vesicle preparation include; Tdfl, NspA, Hsf, Hap, OMP85, TbpA (high), TbpA (low), LbpA, TbpB, LbpB, PilQ, AspA, Tdfl, TdfH, PorB, HpuB, P2086, NM-ADPRT, MafA, MafB and PldA
  • Such preparations may optionally also comprise either or both of LPS immunotype L2 and LPS immunotype L3.
  • the OMV might comprise antigen which have been down-regulated.
  • the manufacture of bleb preparations from Neisserial strains may be achieved by any of the methods well known to a skilled person.
  • OMVs are extracted with a detergent, suitably deoxycholate, and nucleic acids are optionally removed enzymatically. Purification is achieved by ultracentrifugation optionally followed by size exclusion chromatography. If 2 or more different blebs of the disclosure are included, they may be combined in a single container to form a multivalent preparation of the disclosure (although a preparation is also considered multivalent if the different blebs of the disclosure are separate compositions in separate containers which are administered at the same time [the same visit to a practitioner] to a host).
  • OMV preparations are usually sterilised by filtration through a 0.2 Fm filter, and are suitably stored in a sucrose solution (e.g. 3%) which is known to stabilise the bleb preparations.
  • Upregulation of proteins within outer membrane vesicle preparations may be achieved by insertion of an extra copy of a gene into the Neisserial strain from which the OMV preparation is derived.
  • the promoter of a gene can be exchanged for a stronger promoter in the Neisserial strain from which the OMV preparation is derived.
  • Such techniques are described in WO01/09350.
  • Upregulation of a protein will lead to a higher level of protein being present in OMV compared to the level of protein present in OMV derived from unmodified N. meningitidis (for instance strain H44/76). In one aspect the level will be 1.5, 2, 3, 4, 5, 7, 10 or 20 times higher.
  • a protocol using a low concentration of extracting detergent may suitably be used in the OMV preparation method so as to preserve high levels of bound LPS whilst removing particularly toxic, poorly bound LPS.
  • concentration of DOC used is suitably 0-0.5% DOC, 0.02-0.4% DOC, 0.04-0.3% DOC more suitably 0.06%-0.2% DOC or 0.08-0. 15% DOC most suitably around or exactly 0.1% DOC.
  • OMVs may include native OMVs obtained without detergent extraction, suitably over-expressing an antigen such as fHbp.
  • “Stronger promoter sequence” refers to a regulatory control element that increases transcription for a gene encoding antigen of interest.
  • Upregulating expression refers to any means to enhance the expression of an antigen of interest, relative to that of the non-modified (i.e., naturally occurring) bleb.
  • upregulation of an antigen refers to expression that is at least 10% higher than that of the non-modified bleb. In one aspect it is at least 50% higher. More suitably it is at least 100% (2 fold) higher. Most suitably it is 3, 4, 5, 7, 10, 20 fold higher.
  • upregulating expression may refer to rendering expression non-conditional on metabolic or nutritional changes, particularly in the case of TbpA, TbpB, LbpA and LbpB. In one aspect the level of expression is assessed when blebs have been derived from bacteria grown in iron limited conditions (for instance in the presence of an iron chelator).
  • the term “engineering a bacterial strain to produce less of said antigen” or “down regulation” refers to any means to reduce the expression of an antigen (or the expression of a functional gene product) of interest, relative to that of the non-modified (i.e. naturally occurring bleb), suitably by deletion, such that expression is at least 10% lower than that of the non-modified bleb.
  • the down regulated protein is an enzyme or a functional protein
  • the downregulation may be achieved by introducing one or more mutations resulting in a 10%, 20%, 50%, 80% or suitably a 100% reduction in enzymatic or functional activity.
  • Neisserial proteins can be carried out in a variety of ways known to the skilled person. For instance, sequences (e.g. promoters or open reading frames) can be inserted, and promoters/genes can be disrupted by the technique of transposon insertion. For instance, for upregulating a gene's expression, a strong promoter could be inserted via a transposon up to 2 kb upstream of the gene's initiation codon (more suitably 200-600 bp upstream, most suitably approximately 400 bp upstream). Point mutation or deletion may also be used (particularly for down-regulating expression of a gene).
  • sequences e.g. promoters or open reading frames
  • promoters/genes can be disrupted by the technique of transposon insertion.
  • a strong promoter could be inserted via a transposon up to 2 kb upstream of the gene's initiation codon (more suitably 200-600 bp upstream, most suitably approximately 400 bp upstream).
  • the engineering step may be suitably performed via a homologous recombination event.
  • the event takes place between a sequence (a recombinogenic region) of at least 30 nucleotides on the bacterial chromosome, and a sequence (a second recombinogenic region) of at least 30 nucleotides on a vector transformed within the strain.
  • the regions are 40-1000 nucleotides, more suitably 100-800 nucleotides, most suitably 500 nucleotides). These recombinogenic regions should be sufficiently similar that they are capable of hybridising to one another under highly stringent conditions.
  • Typical strong promoters that may be integrated in Neisseria are porA, porB, IgtF, Opa, p110, Ist, and hpuAB.
  • PorA and PorB are suitable as constitutive, strong promoters. It has been established that the PorB promoter activity is contained in a fragment corresponding to nucleotides-1 to -250 upstream of the initiation codon of porB.
  • the upregulation of some antigens in an outer membrane vesicle preparation of the disclosure is suitably achieved by isolating outer membrane vesicles from a parental strain of Neisseria grown under iron limitation conditions.
  • a low concentration of iron in the medium will result in increased expression of proteins involved in iron acquisition including TbpA, TbpB, LbpA, LbpB, HpuA, HpuB and P2086.
  • the expression of these proteins is thereby upregulated without the need for recombinantly modifying the gene involved, for instance by inserting a stronger promoter or inserting an additional copy of the gene.
  • the disclosure would also encompass upregulation of iron acquisition proteins by growth in iron limitation medium where the gene has also been recombinantly modified.
  • Iron limitation is achieved by the addition of an iron chelator to the culture medium.
  • Suitable iron chelators include 2,2-Dipyridil, EDDHA (ethylenediamine-di(o-hydroxyphenylacetic acid) and Desferal (deferoxamine mesylate, Sigma).
  • Desferal is the suitable iron chelator and is added to the culture medium at a concentration of between 10 and 100 pM, suitably 25-75 uM, more suitably 50-70 uM, most suitably at 6011M.
  • the iron content of medium comes primarily from the yeast extract and soy peptone constituents and the amount present may vary between batches. Therefore different concentrations of Desferal may be optimal to achieve upregulation of iron acquisition proteins in different batches of medium. The skilled artisan should easily be able to determine the optimal concentration. In basic terms, enough iron chelator should be added to the medium to upregulate the expression of the desired iron-regulated protein, but not so much so as to adversely affect the growth of the bacteria.
  • upregulation of iron acquisition proteins by growth under iron limited conditions is combined with recombinant upregulation of other antigens so that the outer membrane vesicle of the disclosure is achieved.
  • zinc limitation may be used to increase expression of Tdfl. This may be achieved for example, using a medium low in Zn 2+ concentration—i.e. under 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05 or 0.01 ⁇ M free Zn 2+ —(such as Roswell Park Memorial Institute medium 1640 (RPMI) which has around 1.69 ⁇ M Zn 2+ by ICP-MS), or by removing Zn 2+ in the medium, for instance using a known zinc chelator such as TPEN (N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine)—enough should be added to the medium such that the expression of the NMB0964 is maximised.
  • RPMI Roswell Park Memorial Institute medium 1640
  • TPEN N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine
  • Synthetic media such as a or Catlin adapted media may also be used (Nutritional profiles of Neisseria gonorrhoeae, Neisseria meningitidis , and Neisseria lactamica in chemically defined media and the use of growth requirements for gonococcal typing. Catlin B W J Infect Dis. 1973 August; 128(2):178-94.).
  • OMV vaccine prepared either in specific culture conditions low in Zn2+, or from a mutant N. meningitidis strain engineered to either over-express NMB0964 or to remove the Zinc repression mechanism mediated through Zn2+, is enriched in NMB0964, and such OMVs may elicit good bactericidal antibody responses compared to OMVs which have not been prepared with these methods.
  • variable antigens are variable among bacterial strains and as a consequence are protective only against a limited set of closely related strains.
  • An aspect of this disclosure covers outer membrane vesicles of the disclosure in which the expression of other proteins is reduced, or, suitably, gene (s) encoding variable surface protein (s) are deleted. Such deletion results in a bacterial strain producing blebs which, when administered in a composition or vaccine, have a stronger potential for cross-reactivity against various strains due to a higher influence exerted by conserved proteins (retained on the outer membranes) on the vaccinee's immune system.
  • variable antigens in Neisseria that may be downregulated in the bleb immunogenic compositions of the disclosure include PorA, PorB, Opa.
  • genes which, in vivo, can easily be switched on (expressed) or off by the bacterium are genes which, in vivo, can easily be switched on (expressed) or off by the bacterium.
  • outer membrane proteins encoded by such genes are not always present on the bacteria, the presence of such proteins in the bleb preparations can also be detrimental to the effectiveness of the composition or vaccine for the reasons stated above.
  • a suitable example to down-regulate or delete is Neisseria Opc protein. Anti-Opc immunity induced by an Opc containing bleb vaccine would only have limited protective capacity as the infecting organism could easily become Opc.
  • variable or non-protective genes may be down-regulated in expression, or terminally switched off. This has the advantage of concentrating the immune system on better antigens that are present in low amounts on the outer surface of blebs.
  • down-regulation it is also meant that surface exposed, variable immunodominant loops of the above outer membrane proteins may be altered or deleted in order to make the resulting outer membrane protein less immunodominant.
  • Suitable proteins to be downregulated in the bleb immunogenic compositions of the disclosure include PorA and OpA; PorA and OpC; OpA and OpC; PorA and OpA and OpC.
  • Opa is said to be downregulated in expression it is meant that suitably 1, 2, 3 or (suitably) all 4 genes present in meningococcus are so downregulated.
  • Such downregulation may be performed genetically as described in WO 01/09350 or by seeking readily-found, natural, stable meningococcal strains that have no or low expression from the Opa loci. Such strains can be found using the technique described in Poolman et al (1985 J. Med. Micro.
  • variable protein FrpB (Microbiology 142; 3269-3274, (1996); J. Bacteriol. 181; 2895-2901 (1999)) will also be upregulated.
  • the inventors have found that it is advantageous to downregulate expression of FrpB under these circumstances by downregulating expression of the entire protein as described in WO01/09350 or by deleting variable region (s) of FrpB.
  • FrpB Down regulation of FrpB is suitably combined with down regulation of PorA and OpA; PorA and OpC; OpA and OpC; PorA and OpA and OpC in the bleb immunogenic compositions of the disclosure.
  • FrpB is downregulated in outer membrane vesicles which have been prepared from Neisseria strains not grown under iron limitation conditions.
  • the blebs in the immunogenic compositions of the disclosure may be detoxified via methods for detoxification of LPS which are disclosed in WO01/09350.
  • methods for detoxification of LPS of the disclosure involve the downregulation/deletion of htrB and/or msbB enzymes which are disclosed in WO01/09350.
  • the msbB and htrB genes of Neisseria are also called IpxL1 and IpxL2, respectively (WO 00/26384) and deletion mutations of these genes are characterised pnenoltypically by the msbB-mutant LOS losing one secondary acyl chain), and the htrB-mutatn LOS losing both secondary acyl chains.
  • WO93/14155 and WO 95/03327 describe nontoxic peptide functional equivalents of polymycin B that may be used in compositions of the disclosure.
  • Such methods are suitably combined with methods of bleb extraction involving low levels of DOC, suitably 0-0.3% DOC, more suitably 0.05%-0.2% DOC, most suitably around or exactly 0.1% DOC.
  • LPS detoxification include adding to the bleb preparations a non-toxic peptide functional equivalent of polymyxin B (suitably SAEP 2) as described above.
  • Cross-reactive polysaccharides the isolation of bacterial outer-membrane blebs from encapsulated Gram-negative bacteria often results in the co-purification of capsular polysaccharide.
  • this “contaminant” material may prove useful since polysaccharide may enhance the immune response conferred by other bleb components.
  • the presence of contaminating polysaccharide material in bacterial bleb preparations may prove detrimental to the use of the blebs in a composition or vaccine. For instance, it has been shown at least in the case of N. meningitidis that the serogroup B capsular polysaccharide does not confer protective immunity and is susceptible to induce an adverse auto-immune response in humans.
  • outer membrane vesicles of the disclosure may be isolated from a bacterial strain for bleb production, which has been engineered such that it is free of capsular polysaccharide.
  • the blebs will then be suitable for use in humans.
  • a particularly suitable example of such a bleb preparation is one from N. meningitidis serogroup B devoid of capsular polysaccharide.
  • Inactivation of the gene coding for capsular polysaccharide biosynthesis or export can be achieved by mutating (point mutation, deletion or insertion) either the control region, the coding region or both (suitably using the homologous recombination techniques described above), or by any other way of decreasing the enzymatic function of such genes.
  • inactivation of capsular biosynthesis genes may also be achieved by antisense over-expression or transposon mutagenesis.
  • a suitable method is the deletion of some or all of the Neisseria meningitidis cps genes required for polysaccharide biosynthesis and export.
  • the replacement plasmid pMF121 (described in Frosh et al. 1990, Mol. Microbiol. 4: 1215-1218) can be used to deliver a mutation deleting the cpsCAD (+galE) gene cluster.
  • WO 01/09350 discloses plasmid pMF121 (Frosch et al., 1990) has been used to construct a Neisseria meningitidis B strain lacking the capsular polysaccharide.
  • This plasmid contains the flanking regions of the gene locus coding for the biosynthesis pathway of the group B polysaccharide (B PS), and the erythromycin resistance gene. Deletion of the B PS resulted in loss of expression of the group B capsular polysaccharide as well as a deletion in the active copy of galE leading to the synthesis of galactose deficient LPS.
  • the deletion of the terminal part of the LOS saccharidic is advantageous in preventing any cross-reaction with structures present at the surface of human tissues.
  • inactivation of the IgtB gene results in an intermediate LPS structure in which the terminal galactose residue and the sialic acid are absent (the mutation leaves a 4GlcNAcpl-3Galpl-4Glcpl-structure in L2 and L3 LOS).
  • Such intermediates could be obtained in an L3 and an L2 LPS strain.
  • An alternative and less suitable (short) version of the LPS can be obtained by turning off the IgtE gene.
  • a further alternative is use of the galE mutation ( Cloning and molecular analysis of the galE gene of Neisseria meningitidis and its role in lipopolysaccharide biosynthesis . Jennings M P, van der Ley P, Wilks K E, Maskell D J, Poolman J T, Moxon E R. Mol Microbiol. 1993 October; 10(2):361-9) to obtain both a capsule minus strain and a LOS with a short alpha-chain (as IgtE mutation).
  • a further alternative and less suitable version of the LPS can be obtained by turning off the IgtA gene. If such an IgtA-mutation is selected it is suitable to also turn off IgtC expression to prevent the non-immunogenic L1 immunotype being formed.
  • LgtB-mutants are most suitable as the inventors have found that this is the optimal troncation for resolving the safety issue whilst still retaining an LPS protective oligosaccharide epitope that can still induce a bactericidal antibody response.
  • immunogenic compositions of the disclosure further comprising L2 or L3 preparations (whether purified or in an isolated bleb) or meningococcal bleb preparations in general are advantageously derived from a Neisserial strain (suitably meningococcal) that has been genetic engineered to permanently downregulate the expression of functional gene product from the IgtB, IgtA or IgtE gene, suitably by switching the gene off, most suitably by deleting all or part of the promoter and/or open-reading frame of the gene.
  • a Neisserial strain suitably meningococcal
  • the capsular polysaccharide which also contains human-like saccharide structures
  • the inventors have advantageously shown that it is suitable that the bleb production strain has been genetically engineered to permanently downregulate the expression of functional gene product from the siaD gene (i.e. downregulating a-2-8 polysialyltransferase activity), suitably by switching the gene off, most suitably by deleting all or part of the promoter and/or open-reading frame of the gene.
  • Such an inactivation is described in WO 01/09350.
  • the siaD (also known as synD) mutation is the most advantageous of many mutations that can result in removing the human-similar epitope from the capsular polysaccharide, because it one of the only mutations that has no effect on the biosynthesis of the protective epitopes of LOS, thus being advantageous in a process which aims at ultimately using LOS as a protective antigen, and has a minimal effect on the growth of the bacterium.
  • a suitable aspect of the disclosure is therefore a bleb immunogenic preparation as described above which is derived from an IgtE′siaD′, an IgtA-siaD′ or, suitably, an IgtB-siaD-meningococcus B mutant strain.
  • the strain itself is a further aspect of the disclosure.
  • bleb production strain can be genetically engineered to permanently downregulate the expression of functional gene product from one or more of the following genes: ctrA, ctrB, ctrC, ctrD, synA (equivalent to synX and siaA), synB (equivalent to siaB) or synC (equivalent to siaC) genes, suitably by switching the gene off, most suitably by deleting all or part of the promoter and/or open-reading frame of the gene.
  • the IgtE-mutation may be combined with one or more of these mutations. In one aspect the IgtB-mutation is combined with one or more of these mutations.
  • a further aspect of the disclosure is therefore a bleb immunogenic preparation as described above which is derived from such a combined mutant strain of meningococcus B.
  • the strain itself is a further aspect of the disclosure.
  • the galE mutation may also be used to get both a capsule minus strain and a LOS with a short alpha-chain (as IgtE mutation).
  • LOS In such case, a capsule negative strain which has a deleted synA (equivalent to synX and siaA), synB (equivalent to siaB) or synC (equivalent to siaC) gene is advantageous, as such a mutation also renders menB LOS incapable of being sialylated.
  • LPS may be used as an antigen in the immunogenic composition of the disclosure. It is however advantageous to downregulate/delete/inactivate enzymatic function of either the IgtE, IgtA (particularly in combination with IgtC), or, suitably, IgtB genes/gene products in order to remove human like lacto-N-neotetraose structures.
  • the Neisserial locus (and sequence thereof) comprising the Igt genes for the biosynthesis of LPS oligosaccharide structure is known in the art (Jennings et al Microbiology 1999 145; 3013-3021 and references cited therein, and J. Exp. Med. ⁇ BR> ⁇ BR> ⁇ BR> ⁇ P> 180: 2181-2190) [1994].
  • Downregulation/deletion of IgtB (or functional gene product) is suitable since it leaves the LPS protective epitope intact.
  • N. meningitidis serogroup B bleb preparations of the disclosure the downregulation/deletion of both siaD and IgtB is suitable, (although a combination of IgtB- with any of ctrA-, ctrB-, ctrC-, ctrD′, synA- (equivalent to synX- and siaA-), synB- (equivalent to siam-) or synC- (equivalent to siam-) in a meningococcus B bleb production strain may also be used) leading to a bleb preparation with optimal safety and LPS protective epitope retention.
  • a further aspect of the disclosure is therefore a bleb immunogenic preparation as described above which is derived from such a combined mutant strain of meningococcus B.
  • the strain itself is a further aspect of the disclosure.
  • Immunogenic composition of the disclosure may comprise at least, one, two, three, four or five different outer membrane vesicle preparations. Where two or more OMV preparations are included, at least one antigen of the disclosure is upregulated in each OMV.
  • OMV preparations may be derived from Neisserial strains of the same species and serogroup or suitably from Neisserial strains of different class, serogroup, serotype, subserotype or immunotype.
  • an immunogenic composition may comprise one or more outer membrane vesicle preparation (s) which contains LPS of immunotype L2 and one or more outer membrane vesicle preparation which contains LPS of immunotype L3.
  • L2 or L3 OMV preparations are suitably derived from a stable strain which has minimal phase variability in the LPS oligosaccharide synthesis gene locus.
  • the immunogenic compositions of the disclosure may also comprise both a subunit composition and an outer membrane vesicle. There are several antigens that are particularly suitable for inclusion in a subunit composition due to their solubility.
  • proteins examples include; Tdfl, FhaB, NspA, passenger domain of Hsf, passenger domain of Hap, passenger domain of AspA, AspA, OMP85, FrpA, FrpC, TbpB, LbpB, PilQ.
  • the outer membrane vesicle preparation would have at least one different antigen selected from the following list which has been recombinantly upregulated in the outer membrane vesicle: NspA, Hsf, Hap, OMP85, TbpA (high), TbpA (low), LbpA, TbpB, LbpB, NadA, TspA, TspB, PilC, PilQ, TdfH, PorB, HpuB, P2086, NM-ADPRT, MafA, MafB and PIdA; and optionally comprise either or both of LPS immunotype L2 and LPS immunotype L3.
  • Another aspect of the disclosure is a genetically engineered Neisserial strain from which an outer membrane vesicle of the disclosures (optionally having at least two proteins of the disclosure recombinantly upregulated, as described above) may be derived.
  • Such Neisserial strains may be Neisseria meningitidis or Neisseria gonorrhoeae.
  • the strain may also have been engineered (as described above) to downregulate the expression of other Neisserial proteins including the expression of one, two, three, four, five, six, seven or eight of LgtB, LgtE, SiaD, OpC, OpA, PorA, FrpB, msbB and HtrB.
  • Suitable combinations for downregulation include down regulation (suitably deletion) of at least LgtB and SiaD, downregulation of at least PorA and OpC, downregulation of at least PorA and OpA and downregulation of at least PorA, OpA and OpC.
  • further aspects of the disclosure includes methods of making the immunogenic composition or vaccine of the disclosure. These include a method comprising a step of mixing together the chimaeric protein of the disclosure with an isolated antigens or proteins from Neisseria , which may be present in the form of blebs derived from the Neisserial strains of the disclosure, to make an immunogenic composition of the disclosure, and a method of making the composition or vaccine of the disclosure comprising a step of combining the immunogenic composition of the disclosure with a pharmaceutically acceptable carrier.
  • Also included in the disclosure are methods of making the immunogenic composition of the disclosure comprising a step of isolating outer membrane vesicles comprising the chimaeric protein from a Neisserial culture.
  • Such a method may involve a further step of combining at least two outer membrane vesicle preparations, in one aspect wherein at least one outer membrane vesicle preparation contains LPS of immunotype L2 and at least one outer membrane vesicle preparation contains LPS of immunotype L3.
  • the disclosure also includes such methods wherein the outer membrane vesicles are isolated by extracting with a concentration of DOC of 0-0.5%. DOC concentrations of 0.3%-0.5% are used to minimise LPS content. In OMV preparations where LPS is to be conserved as an antigen, DOC concentrations of 0-0.3%, suitably 0.05%-0.2%, most suitably of about 0.1% are used for extraction.
  • the immunogenic composition of the disclosure may further comprise bacterial capsular polysaccharides or oligosaccharides.
  • the capsular polysaccharides or oligosaccharides may be derived from one or more of: Neisseria meningitidis serogroup A, C, Y, and/or W-135 , Haemophilus influenzae b, Streptococcus pneumonia , Group A Streptococci, Group B Streptococci, Staphylococcus aureus and Staphylococcus epidermidis.
  • composition or vaccine combinations comprising the antigenic composition of the disclosure with other antigens which are advantageously used against certain disease states including those associated with viral or Gram positive bacteria.
  • the antigenic compositions of the disclosure are formulated with 1, 2, 3 or suitably all 4 of the following meningococcal capsular polysaccharides or oligosaccharides which may be plain or conjugated to a protein carrier: A, C, Y or W-135.
  • the immunogenic compositions of the disclosure are formulated with A and C; or C; or C and Y.
  • Such a composition or vaccine containing proteins from N. meningitidis , suitably serogroup B may be advantageously used as a global meningococcus vaccine.
  • the antigenic compositions of the disclosure are formulated with 1, 2, 3 or all 4 of the plain or conjugated meningococcal capsular polysaccharides or oligosaccharides A, C, Y or W-135 (as described above), are formulated with a conjugated H. influenzae b capsular polysaccharide or oligosaccharides, and/or one or more plain or conjugated pneumococcal capsular polysaccharides or oligosaccharides.
  • the vaccine may also comprise one or more protein antigens that can protect a host against Streptococcus pneumoniae infection. Such a vaccine may be advantageously used as a global meningitis vaccine.
  • the immunogenic composition of the disclosure is formulated with capsular polysaccharides or oligosaccharides derived from one or more of Neisseria meningitidis, Haemophilus influenzae b, Streptococcus pneumoniae , Group A Streptococci, Group B Streptococci, Staphylococcus aureus or Staphylococcus epidermidis .
  • the pneumococcal capsular polysaccharide antigens are suitably selected from serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F (most suitably from serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F).
  • a further suitable embodiment would contain the PRP capsular polysaccharides of Haemophilus influenzae .
  • a further suitable embodiment would contain the Type 5, Type 8 or 336 capsular polysaccharides of Staphylococcus aureus .
  • a further suitable embodiment would contain the Type I, Type II or Type III capsular polysaccharides of Staphylococcus epidermidis .
  • a further suitable embodiment would contain the Type Ia, Type Ic, Type II or Type III capsular polysaccharides of Group B streptococcus .
  • a further suitable embodiment would contain the capsular polysaccharides of Group A streptococcus , suitably further comprising at least one M protein and more suitably multiple types of M protein.
  • Such capsular polysaccharides of the disclosure may be unconjugated or conjugated to a carrier protein such as tetatus toxoid, tetanus toxoid fragment C, diphtheria toxoid, CRM197, pneumolysin, Protein D (U.S. Pat. No. 6,342,224).
  • the polysaccharide conjugate may be prepared by any known coupling technique.
  • the polysaccharide can be coupled via a thioether linkage.
  • This conjugation method relies on activation of the polysaccharide with 1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) to form a cyanate ester.
  • CDAP 1-cyano-4-dimethylamino pyridinium tetrafluoroborate
  • the activated polysaccharide may thus be coupled directly or via a spacer group to an amino group on the carrier protein.
  • the cyanate ester is coupled with hexane diamine and the amino-derivatised polysaccharide is conjugated to the carrier protein using heteroligation chemistry involving the formation of the thioether linkage.
  • Such conjugates are described in PCT published application WO93/15760 Uniformed Services University.
  • the conjugates can also be prepared by direct reductive amination methods as described in U.S. Pat. No. 4,365,170 (Jennings) and U.S. Pat. No. 4,673,574 (Anderson). Other methods are described in EP-0-161-188, EP-208375 and EP-0-477508.
  • a further method involves the coupling of a cyanogen bromide activated polysaccharide derivatised with adipic acid hydrazide (ADH) to the protein carrier by Carbodiimide condensation (Chu C. et al Infect. Immunity, 1983 245 256). Where oligosaccharides are included, it is suitable that they be conjugated.
  • Suitable pneumococcal proteins antigens are those pneumococcal proteins which are exposed on the outer surface of the pneumococcus (capable of being recognised by a host's immune system during at least part of the life cycle of the pneumococcus), or are proteins which are secreted or released by the pneumococcus.
  • the protein is a toxin, adhesin, 2-component signal tranducer, or lipoprotein of Streptococcus pneumoniae , or fragments thereof.
  • Particularly suitable proteins include, but are not limited to: pneumolysin (suitably detoxified by chemical treatment or mutation) [Mitchell et al. Nucleic Acids Res. 1990 Jul. 11; 18 (13): 4010 “Comparison of pneumolysin genes and proteins from Streptococcus pneumoniae types 1 and 2.”, Mitchell et al. Biochim Biophys Acta 1989 Jan.
  • pneumococcal protein antigens are those disclosed in WO 98/18931, particularly those selected in WO 98/18930 and PCT/US99/30390.
  • the immunogenic composition/vaccine of the disclosure may also optionally comprise outer membrane vesicle preparations made from other Gram negative bacteria, for example Moraxella catarrhalis or Haemophilus influenzae.
  • Immunogenic compositions of the disclosure may further comprise OMV preparations derived from Moraxella catarrhalis .
  • Engineered OMV preparations can be derived from Moraxella catarrhalis as described in WO01/09350.
  • One or more of the following genes (encoding protective antigens) are suitable for upregulation: OMP106 (WO 97/41731 & WO 96/34960), HasR (PCT/EP99/03824), PilQ (PCT/EP99/03823), OMP85 (PCT/EP00/01468), lipo06 (GB 9917977.2), lipolO (GB 9918208.1), lipoll (GB 9918302.2), lipol8 (GB 9918038.2), P6 (PCT/EP99/03038), ompCD, CopB (Helminen M E, et al (1993) Infect.
  • One or more of the following genes are suitable for downregulation: CopB, OMP106, OmpBl, TbpA, TbpB, LbpA, and LbpB.
  • One or more of the following genes are suitable for downregulation: htrB, msbB and IpxK.
  • One or more of the following genes are suitable for upregulation: pmrA, pmrB, pmrE, and pmrF.
  • Immunogenic compositions of the disclosure may further comprise OMV preparations derived from Haemophilus influenzae .
  • Engineered OMV preparations can be derived from Haemophilus influenzae as described in WO01/09350.
  • One or more of the following genes (encoding protective antigens) are suitable for upregulation: D15 (WO 94/12641), P6 (EP 281673), TbpA (WO96/40929; WO95/13370), TbpB (WO96/40929; WO95/13370), P2, P5 (WO 94/26304), OMP26 (WO 97/01638), HMW1, HMW2, HMW3, HMW4, Hia, Hsf, Hap, Hin47, and Hif (all genes in this operon should be upregulated in order to upregulate pilin). They are also suitable as genes which may be heterologously introduced into other Gram-negative bacteria.
  • One or more of the following genes are preferred for downregulation: P2, P5, Hif, IgAl-protease, HgpA, HgpB, HMW1, HMW2, Hxu, htrB, msbB and IpxK.
  • pmrA pmrB, pmrE, and pmrF.
  • the immunogenic composition/vaccine of the disclosure may also optionally comprise antigens providing protection against one or more of Diphtheria, tetanus and Bordetella pertussis infections.
  • the pertussis component may be killed whole cell B. pertussis (Pw) or acellular pertussis (Pa) which contains at least one antigen (suitably 2 or all 3) from PT, FHA and 69 kDa pertactin.
  • the antigens providing protection against Diphtheria and tetanus would be Diphtheria toxoid and tetanus toxoid.
  • the toxoids may chemically inactivated toxins or toxins inactivated by the introduction of point mutations.
  • the immunogenic composition/vaccine may also optionally comprise one or more antigens that can protect a host against non-typeable Haemophillus influenzae , RSV and/or one or more antigens that can protect a host against influenza virus.
  • a vaccine may be advantageously used as a global otitis media vaccine.
  • Suitable non-typeable H. influenzae protein antigens include Fimbrin protein (U.S. Pat. No. 5,766,608) and fusions comprising peptides therefrom (eg LB1 Fusion) (U.S. Pat. No. 5,843,464-Ohio State Research Foundation), OMP26, P6, protein D, TbpA, TbpB, Hia, Hmwl, Hmw2, Hap, and D15.
  • Suitable influenza virus antigens include whole, live or inactivated virus, split influenza virus, grown in eggs or MDCK cells, or Vero cells or whole flu virosomes (as described by R. Gluck, Vaccine, 1992, 10, 915-920) or purified or recombinant proteins thereof, such as HA, NP, NA, or M proteins, or combinations thereof.
  • Suitable RSV (Respiratory Syncytial Virus) antigens include the F glycoprotein, the G glycoprotein, the HN protein, the M protein or derivatives thereof.
  • Immunogenic compositions of the disclosure may include proteins of Moraxella catarrhalis include TbpA (WO97/13785; WO99/52947), TbpB (WO97/13785; WO99/52947; Mathers et al FEMS Immunol Med Microbiol 1997 19; 231-236; Myers et al Infect Immun 1998 66; 4183-4192), LbpA, LbpB (Du et al Infect Immun 1998 66; 3656-3665), UspAl, UspA2 (Aebi et al Infect Immun. 1997 65; 4367-4377), OMP106 (U.S. Pat. No.
  • the disclosure also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising antigens and immunogenic compositions of the disclosure in combination with a pharmaceutically acceptable excipient.
  • Suitable excipients are well known in the art. Suitable excipients are typically large, slowly metabolised macromolecules such as proteins, saccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, sucrose (Paoletti et al., 2001, Vaccine, 19:2118), trehalose (WO 00/56365), lactose and lipid aggregates (such as oil droplets or liposomes). Such carriers are well known to those of ordinary skill in the art.
  • the vaccines may also contain diluents, such as water, saline, glycerol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present. Sterile pyrogen-free, phosphate buffered physiologic saline is a typical carrier. A thorough discussion of pharmaceutically acceptable excipients is available in reference Gennaro, 2000, Remington: The Science and Practice of Pharmacy, 20.sup.th edition, ISBN:0683306472.
  • the disclosure relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an fHbp polypeptide antigen and a second antigen capable of generating an antibody response against a Neisserial meningitidis L2 immunotype, in combination with a pharmaceutically acceptable excipient.
  • One embodiment of the disclosure is the formulation of the antigens and immunogenic compositions of the disclosure in a vaccine a pharmaceutically acceptable excipient.
  • Vaccine preparation is generally described in Vaccine Design (“The subunit and adjuvant approach” (eds Powell M. F. & Newman M. J.) (1995) Plenum Press New York).
  • Serum Bactericidal Activity (SBA) assays may be used to determine suitable antigens for inclusion in any vaccine, and suitably a four-fold increase in SBA may be accepted as a surrogate for protection.
  • the vaccines or immunogenic compositions of the invention may comprise a human dose of (or of more than) 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ⁇ g of each of the recited (isolated and/or purified) antigens in the composition.
  • a pharmaceutical composition or vaccine of the disclosure may also comprise an adjuvant.
  • Suitable adjuvants include an aluminium salt such as aluminum hydroxide gel (alum) or aluminium phosphate, but may also be a salt of calcium (particularly calcium carbonate), iron or zinc, or may be an insoluble suspension of acylated tyrosine, or acylated sugars, cationically or anionically derivatised polysaccharides, or polyphosphazenes.
  • aluminium salt such as aluminum hydroxide gel (alum) or aluminium phosphate
  • alum aluminum hydroxide gel
  • aluminium phosphate but may also be a salt of calcium (particularly calcium carbonate), iron or zinc, or may be an insoluble suspension of acylated tyrosine, or acylated sugars, cationically or anionically derivatised polysaccharides, or polyphosphazenes.
  • Thl adjuvant systems that may be used include, Monophosphoryl lipid A, particularly 3-de-O-acylated monophosphoryl lipid A, and a combination of monophosphoryl lipid A, suitably 3-de-O-acylated monophosphoryl lipid A (3D-MPL) together with an aluminium salt (suitably aluminium phosphate).
  • An enhanced system involves the combination of a monophosphoryl lipid A and a saponin derivative particularly the combination of QS21 and 3D-MPL as disclosed in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol as disclosed in WO96/33739.
  • a particularly potent adjuvant formulation involving QS21 3D-MPL and tocopherol in an oil in water emulsion is described in WO95/17210 and is a suitable formulation.
  • the vaccine may comprise a saponin, more suitably QS21. It may also comprise an oil in water emulsion and tocopherol. Unmethylated CpG containing oligo nucleotides (WO 96/02555) are also preferential inducers of a TH1 response and are suitable for use in the present disclosure.
  • Another aspect of the disclosure is a method of preparing an immunoglobulin for use in prevention or treatment of Neisserial infection comprising the steps of immunizing a recipient with the vaccine of the disclosure and isolating immunoglobulin from the recipient.
  • An immunoglobulin prepared by this method is a further aspect of the disclosure.
  • a pharmaceutical composition comprising the immunoglobulin of the disclosure and a pharmaceutically acceptable carrier is a further aspect of the disclosure which could be used in the manufacture of a medicament for the treatment or prevention of Neisserial disease.
  • a method for treatment or prevention of Neisserial infection comprising a step of administering to a patient an effective amount of the pharmaceutical preparation of the disclosure is a further aspect of the disclosure.
  • Inocula for polyclonal antibody production are typically prepared by dispersing the antigenic composition in a physiologically tolerable diluent such as saline or other adjuvants suitable for human use to form an aqueous composition.
  • a physiologically tolerable diluent such as saline or other adjuvants suitable for human use to form an aqueous composition.
  • An immunostimulatory amount of inoculum is administered to a mammal and the inoculated mammal is then maintained for a time sufficient for the antigenic composition to induce protective antibodies.
  • the antibodies can be isolated to the extent desired by well known techniques such as affinity chromatography (Harlow and Lane Antibodies; a laboratory manual 1988).
  • Antibodies can include antiserum preparations from a variety of commonly used animals e.g. goats, primates, donkeys, swine, horses, guinea pigs, rats or man. The animals are bled and serum recovered.
  • An immune globulin produced in accordance with the present disclosure can include whole antibodies, antibody fragments or subfragments.
  • Antibodies can be whole immunoglobulins of any class e.g. IgG, IgM, IgA, IgD or IgE, chimeric antibodies or hybrid antibodies with dual specificity to two or more antigens of the disclosure. They may also be fragments e.g. F (ab′)2, Fab′, Fab, Fv and the like including hybrid fragments.
  • An immune globulin also includes natural, synthetic or genetically engineered proteins that act like an antibody by binding to specific antigens to form a complex.
  • a vaccine of the present disclosure can be administered to a recipient who then acts as a source of immune globulin, produced in response to challenge from the specific vaccine.
  • a subject thus treated would donate plasma from which hyperimmune globulin would be obtained via conventional plasma fractionation methodology.
  • the hyperimmune globulin would be administered to another subject in order to impart resistance against or treat Neisserial infection.
  • Hyperimmune globulins of the disclosure are particularly useful for treatment or prevention of Neisserial disease in infants, immune compromised individuals or where treatment is required and there is no time for the individual to produce antibodies in response to vaccination.
  • An additional aspect of the disclosure is a pharmaceutical composition
  • a pharmaceutical composition comprising two of more monoclonal antibodies (or fragments thereof; suitably human or humanized) reactive against at least two constituents of the immunogenic composition of the disclosure, which could be used to treat or prevent infection by Gram negative bacteria, suitably Neisseria , more suitably Neisseria meningitidis or Neisseria gonorrhoeae and most suitably Neisseria meningitidis serogroup B.
  • Such pharmaceutical compositions comprise monoclonal antibodies that can be whole immunoglobulins of any class e.g. IgG, IgM, IgA, IgD or IgE, chimeric antibodies or hybrid antibodies with specificity to two or more antigens of the disclosure. They may also be fragments e.g. F (ab′)2, Fab′, Fab, Fv and the like including hybrid fragments.
  • monoclonal antibodies are well known in the art and can include the fusion of splenocytes with myeloma cells (Kohler and Milstein 1975 Nature 256; 495; Antibodies-a laboratory manual Harlow and Lane 1988).
  • monoclonal Fv fragments can be obtained by screening a suitable phage display library (Vaughan T J et al 1998 Nature Biotechnology 16; 535).
  • Monoclonal antibodies may be humanized or part humanized by known methods.
  • Another aspect of the disclosure involves a method for treatment or prevention of Neisserial disease comprising administering a protective dose (or effective amount) of the vaccine of the disclosure to a host in need thereof.
  • the disclosure also includes a use of the immunogenic composition of the disclosure in the preparation of a medicament for treatment or prevention of Neisserial infection or disease, and to an immunogenic composition as described herein for treatment or prevention of Neisserial meningitidis infection or disease.
  • the prevention is prevention against menB infection and/or disease.
  • the host is suitably a human host.
  • the vaccine preparation of the present disclosure may be used to protect or treat a mammal susceptible to infection, by means of administering said vaccine via systemic or mucosal route.
  • administrations may include injection via the intramuscular, intraperitoneal, intradermal or subcutaneous routes; or via mucosal administration to the oral/alimentary, respiratory, genitourinary tracts.
  • one aspect of the present disclosure is a method of immunizing a human host against a disease caused by infection of a gram-negative bacteria, which method comprises administering to the host an immunoprotective dose of the preparation of the present disclosure.
  • each vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccines. Such amount will vary depending upon which specific immunogen is employed and how it is presented. Generally, it is expected that each dose will comprise l-100 pg of protein antigen or OMV preparation, suitably 5-50 pg, and most typically in the range 5-25 ut.
  • An optimal amount for a particular vaccine can be ascertained by standard studies involving observation of appropriate immune responses in subjects.
  • subjects may receive one or several booster immunizations adequately spaced.
  • the vaccines of the disclosure are suitably immunoprotective and non-toxic and suitable for paediatric or adolescent use.
  • paediatric use it is meant use in infants less than 4 years old.
  • immunoprotective it is meant that the SBA and/or animal protection model and/or adhesion blocking assay described above are satisfactorily met.
  • non-toxic it is meant that there is no more than a satisfactory level of endotoxin activity in the vaccine as measured by the well-known LAL and pyrogenicity assays.
  • the efficacy of vaccines can be assessed through a variety of assays. Protection assays in animal models are well known in the art. Furthermore, serum bactericidal assay (SBA) is the most commonly agreed immunological marker to estimate the efficacy of a meningococcal vaccine (Perkins et al. J Infect Dis. 1998, 177: 683-691).
  • SBA serum bactericidal assay
  • Such a synergistic response may be characterised by the SBA elicited by the combination of antigens being at least 50%, two times, three times, suitably four times, five times, six times, seven times, eight times, nine times and most suitably ten times higher than the SBA elicited by each antigen separately.
  • SBA is measured against a homologous strain from which the antigens are derived and suitably also against a panel of heterologous strains.
  • SBA is the most commonly agreed immunological marker to estimate the efficacy of a meningococcal vaccine (Perkins et al. J Infect Dis. 1998, 177: 683-691). Satisfactory SBA can be acertained by any known method. SBA can be carried out using sera obtained from animal models or from human subjects.
  • a suitable method of conducting SBA with human sera is the following.
  • a blood sample is taken prior to the first vaccination, two months after the second vaccination and one month after the third vaccination (three vaccinations in one year being a typical human primary vaccination schedule administered at, for instance, 0, 2 and 4 months, or 0, 1 and 6 months).
  • Such human primary vaccination schedules can be carried out on infants under 1 year old (for instance at the same time as Hib vaccinations are carried out) or 2-4 year olds or adolescents may also be vaccinated to test SBA with such a primary vaccination schedule.
  • a further blood sample may be taken 6 to 12 months after primary vaccination and one month after a booster dose, if applicable.
  • SBA will be satisfactory for an antigen or bleb preparation with homologous bactericidal activity if one month after the third vaccine dose (of the primary vaccination schedule) (in 2-4 year olds or adolescents, but suitably in infants in the first year of life) the percentage of subjects with a four-fold increase in terms of SBA (antibody dilution) titre (compared with pre-vaccination titre) against the strain of meningococcus from which the antigens of the disclosure were derived is greater than 30%, suitably greater than 40%, more suitably greater than 50%, and most suitably greater than 60% of the subjects.
  • an antigen or bleb preparation with heterologous bactericidal activity can also constitute bleb preparation with homologous bactericidal activity if it can also elicit satisfactory SBA against the meningococcal strain from which it is derived.
  • SBA will be satisfactory for an antigen or bleb preparation with heterologous bactericidal activity if one month after the third vaccine dose (of the primary vaccination schedule) (in 2-4 year olds or adolescents, but suitably in infants in the first year of life) the percentage of subjects with a four-fold increase in terms of SBA (antibody dilution) titre (compared with pre-vaccination titre) against three heterologous strains of meningococcus is greater than 20%, suitably greater than 30%, more suitably greater than 35%, and most suitably greater than 40% of the subjects.
  • SBA antibody dilution
  • Such a test is a good indication of whether the antigen or bleb preparation with heterologous bactericidal activity can induce cross-bactericidal antibodies against various meningococcal strains.
  • the three heterologous strains should suitably have different electrophoretic type (ET)-complex or multilocus sequence typing (MLST) pattern (see Maiden et al. PNAS USA 1998, 95: 3140-5) to each other and suitably to the strain from which the antigen or bleb preparation with heterologous bactericidal activity is made or derived.
  • ET electrophoretic type
  • MLST multilocus sequence typing
  • a skilled person will readily be able to determine three strains with different ET-complex which reflect the genetic diversity observed amongst meningococci, particularly amongst meningococcus type B strains that are recognised as being the cause of significant disease burden and/or that represent recognised MenB hyper-virulent lineages (see Maiden et al. supra).
  • strains that could be used are the following: BZ10 (B: 2b: P1.2) belonging to the A-4 cluster; B16B6 (B: 2a: P1.2) belonging to the ET-37 complex; and H44/76 (B: 15: P1.7, 16) belonging to the ET-5 complex, or any other strains belonging to the same ET/Cluster.
  • Such strains may be used for testing an antigen or bleb preparation with heterologous bactericidal activity made or derived from, for instance, meningococcal strain CU385 (B: 4: P1.15) which belongs to the ET-5 complex.
  • Another sample strain that could be used is from the Lineage 3 epidemic clone (e.g. NZ124 [B: 4: P1.7, 4]).
  • Another ET-37 strain is NGP165 (B: 2a: P1.2).
  • TPEN Zinc chelator
  • This can be suspended in a medium with BSA (such as Hanks medium with 0.3% BSA) in order to obtain a working cell suspension adjusted to approximately 20000 CFU/ml.
  • BSA Hanks medium with 0.3% BSA
  • a series of reaction mixes can be made mixing a series of two-fold dilutions of sera to be tested (suitably heat-inactivated at 56° C. for 30 min) [for example in a 50 well volume] and the 20000 CFU/ml meningococcal strain suspension to be tested [for example in a 25 well volume].
  • the reaction vials should be incubated (e.g. 37° C. for 15 minutes) and shaken (e.g. at 210 rpm).
  • the final reaction mixture [for example in a 100 ⁇ l volume] additionally contains a complement source [such as 25% final volume of pretested baby rabbit serum], and is incubated as above [e.g. 37° C. for 60 min].
  • a complement source such as 25% final volume of pretested baby rabbit serum
  • a sterile polystyrene U-bottom 96-well microtiter plate can be used for this assay.
  • a aliquot [e.g. 10 ⁇ l] can be taken from each well using a multichannel pipette, and dropped onto Mueller-Hinton agar plates (suitably containing 1% Isovitalex and 1% heat-inactivated Horse Serum) and incubated (for example for 18 hours at 37° C. in 5% C02).
  • individual colonies can be counted up to 80 CFU per aliquot.
  • test samples can be used as controls: buffer+bacteria+complement; buffer+bacteria+inactivated complement; serum+bacteria+inactivated complement.
  • SBA titers can be straightforwardly calculated using a program which processes the data to give a measurement of the dilution which corresponds to 50% of cell killing by a regression calculation.
  • the synergistic response may be characterised by the efficacy of the combination of anigens in an adhesion blocking assay.
  • the extent of blocking induced by antisera raised against the combination of antigens is significantly improved compared with using antisera raised against the antigens by themselves, particularly at suboptimal doses of antibody.
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
  • Embodiments herein relating to “vaccine compositions” of the disclosure are also applicable to embodiments relating to “immunogenic compositions” of the disclosure, and vice versa.
  • A, B, C, or combinations thereof refers to all permutations and combinations of the listed items preceding the term.
  • A, B, C, or combinations thereof is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.
  • expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, BBC, AAABCCCC, CBBAAA, CABABB, and so forth.
  • BB Broadband
  • AAA AAA
  • BBC AAABCCCCCC
  • CBBAAA CABABB
  • references to blebs, vesicles and outermembrane vesicles herein is also intended to be a reference to all isolated membrane-derived proteinaceous products known to persons of skill in the art such as blebs, microvesicles, OMVs, OMPC (outer membrane protein complex), or membrane ghosts, and the like.
  • compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of suitable embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.
  • fHBPs variants (v) A and B were produced in BL21 (DE3) E. coli after IPTG induction and purification using IMAC column.
  • the fhbp sequences were derived from strains MC58 (fHBP B) and 2996 (fHBP A).
  • Genes were cloned in pET24b plasmid without the nucleotide sequence corresponding to the leader sequence and with a His-Tag in C-ter.
  • Recombinant nadA was also produced in BL21 E. coli and the sequence was derived from strain 2996.
  • mice Groups of 300F1 mice were immunized three times by the intramuscular (IM) route on day 0, 21 and 28 with purified recombinant proteins adsorbed onto Al(OH) 3 . On day 42, blood samples were taken for serum. Mice sera were from experiments 20080232.
  • IM intramuscular
  • N. meningitidis strains were cultivated overnight on MH agar plates at 37° C. +5% CO 2 . They were sub-cultured for 4 hours on MH agar plates with 20 ⁇ M TPEN (zinc chelator) 37° C. +5% CO 2 .
  • Inactivation was performed by incubating the cells harvested from agar plates in PBS-PMSF (200 ⁇ M)-azide (0.2%) ON at 37° C. Inactivated cells were then washed in PBS and frozen at ⁇ 20° C.
  • fHbp allele was determined by PCR typing as described by Beerninck and collaborators in 2006.
  • the complete fhbp locus was amplified from crude MenB lysate by PCR with primers.
  • the PCR fragment was then purified with High Pure PCR Purification Kit (Roche) and sequenced by the Sanger method.
  • the sequence type (family A or B) was deduced after comparison with family A and B reference sequences (2996 and MC58 respectively) using Lasergene MegAlign-ClustalX software as described in Fletcher et al, 2004 (FIG. 2).
  • fHBPs variants (v) A and B were produced in BL21 (DE3) E. coli after IPTG induction and purification using IMAC column.
  • the fHbp sequences were derived from strains MC58 (fHBP B) and 2996 (fHBP A). Genes were cloned in pET24b plasmid without the nucleotide sequence corresponding to the leader sequence and with a His-Tag in C-ter.
  • mice were immunized three times by the intramuscular (1M) route on day 0, 21 and 28.
  • Each injection contained either OMV antigen normalized to 5 ug of protein and formulated with AS04 Adjuvant System (AIP04 plus 3-O-desacyl-4′ monophosphoryl lipid A) or with monovalent fHBP vaccine (fHBP A or fHBP B) adsorbed onto Al(OH) or bivalent fHbpA+B vaccine.
  • AS04 Adjuvant System AS04 plus 3-O-desacyl-4′ monophosphoryl lipid A
  • fHBP A or fHBP B monovalent fHBP vaccine
  • blood samples were taken for serum. Mice sera were from experiments 20040652, 20070371 and 20080083.
  • N. meningitidis strains were cultivated overnight on MH agar plates at 37° C. +5% CO2. They were subcultured for 4 hours on MH agar plates with 20 ⁇ M TPEN (zinc chelator) 37° C.+5% CO2. Inactivation was performed by incubating the cells harvested from agar plates in PBS-PM SF (200 ⁇ M)-azide (0.2%) ON at 37° C. Inactivated cells were then washed in PBS and frozen at ⁇ 20° C.
  • N. meningitidis strains were cultivated overnight on Petri Dishes at 37° C. +5% CO. They were sub-cultured for 4 hours on Petri Dishes without or with 20 ⁇ M TPEN (zinc chelator) 37° C. +5% CO2.
  • Serum samples (pooled sera) were inactivated for 40 min at 56° C. and then diluted 1110 or 1150 in PBS-glucose 0.1% and then twofold diluted in a volume of 25 ⁇ L in flat bottom microplates. Then 25 ⁇ L of a mix of bacteria (diluted in PBS-glucose 0.1% to yield—100-150 CFU per well) and baby rabbit complement (final concentration in microwell: 12.5% v/v) was added to the serum dilution.
  • the SBA titer is the dilution giving 50% of killing.
  • the complete fhbp locus was amplified from crude MenB lysate by PCR with primers.
  • the PCR fragment was then purified with High Pure PCR Purification Kit (Roche) and sequenced by the Sanger method.
  • the sequence type (variant 1, 2 or 3) was deduced after comparison with variant 1, 2 and 3 references sequences using Lasergene MegAlign-ClustalX software.
  • mice were immunized with different OMV preparations obtained from recombinant H44/76 strains having a common background: porA KO, galE LOS and capsule minus.
  • the different preparations are differentiated by the level of Tdfl and fHbp.
  • Control OMVs (Ctrl OMVs) had no detectable amount of Tdfl and fHbp.
  • Tdfl OMVs were produced from a strain that overexpressed Tdfl and Tdfl-fHBPOMVs displayed both Tdfl and fHbp.
  • the sera were analysed in SBA using a panel of H44/76 strains expressing different level of Tdfl.
  • the wild type (WT) strain did not express any detectable amount of Tdfl while a recombinant H44/76 strain transformed with the pfP10 plasmid containing the Tdfl gene under the pTac promoter produced high level of Tdfl in presence of ITPG (IPTG) (Table 4).
  • mice immunized with the control preparation were not bactericidal. Only the strain expressing high amount of Tdfl (IPTG) was killed by anti-Tdfl antibodies in presence of complement.
  • sera from mice immunized with Tdfl-fHbp OMV preparation mediated the complement killing of the two H44/76 strains, via the presence of anti-fHbp antibodies and as observed there was a correlation between the bactericidal titer and the level of Tdfl produced by the targeted strains (Table 4).
  • Outer membranes vesicles were produced using classical 0.5% DOC extraction from different recombinant H44/76 strains (porA KO, capsule minus, galE LOS and over-producing different outer-membrane proteins).
  • fHBPs variants (v) A and B were produced in BL21 (DE3) E. coli after IPTG induction and purification using IMAC column.
  • the fhbp sequences were derived from strains MC58 (fHBP B) and 2996 (fHBP A). Genes were cloned in pET24b plasmid without the nucleotide sequence corresponding to the leader sequence and with a His-Tag in C-ter.
  • mice were immunized three times by the intramuscular (IM) route on day 0, 21 and 28. Each injection contained 5 ⁇ g of monovalent fHBP vaccine (fHBP A or fHBP B) adsorbed onto Al(OH) 3 . On day 42, blood samples were taken for serum. Mice sera were from experiments 20080790 and 20090265.
  • IM intramuscular
  • fHBP A or fHBP B monovalent fHBP vaccine
  • guinea-pigs were immunized three times by the intramuscular (IM) route on day 0, 14 and 28. Each injection contained either OMV antigen normalized to 10 ⁇ g of protein and formulated with AIPO 4 . On day 42, blood samples were taken for serum. Guinea pig sera were from experiments 20090266.
  • IM intramuscular
  • N. meningitidis strains were cultivated overnight on Petri Dishes at 37° C. +5% CO 2 . They were sub-cultured for 4 hours on Petri Dishes without or with 20 ⁇ M TPEN (zinc chelator) 37° C. +5% CO 2 . Serum samples (pooled sera) were inactivated for 40 min at 56° C. and then diluted 1/10 or 1/50 in PBS-glucose 0.1% and then twofold diluted in a volume of 25 ⁇ l in flat bottom microplates.
  • Anti-fHbpB sera were tested in SBA with strains expressing the fHbp family B, while anti-fHbpA sera were used in SBA against strains expressing the fHbp from family A.
  • Sera from guinea-pigs immunized with OMVs (blebs) produced from a strain over-expressing Tdfl were tested against both fHbpB and fHbpA strains.
  • Anti-Tdfl and anti-fHbp sera were tested alone or were mixed before to perform SBA in presence or absence of TPEN.
  • a panel of 16 strains was used in SBA. Among those, 5 to 7 were killed by anti-fHbp antibodies (pending the absence or presence of TPEN in SBA culture condition) (SBA titer >128). In absence of TPEN, only 3 strains were killed by sera from guinea-pig immunized with Tdfl-blebs while in presence of TPEN, 12 strains were killed.
  • T7 Express competent E. coli (NEB catalogue number C2566H): Enhanced BL21 derivative.
  • B Strain Free of animal products.
  • the Family B part of the fusion exemplified herein starts from amino acid position 73 of the full length MC58 sequence, which full length sequence itself comprises a mature sequence starting at amino acid 66.
  • the 7 N-ter aa (CSSGGGG—SEQ ID NO. 13) are replaced by MHHHHHH—SEQ ID NO. 14 to allow purification).
  • MC58 part of the fusion finishes at residue 200 ( . . . DIA) if we take account the numbering of the full length MC58 protein sequence, with residue 200 of the full length sequence corresponds to the residue 135 of the mature MC58 sequence.
  • the 8047 part of the fusion exemplified herein begins at residue 155 of the full length (273 amino acid) 8047 protein sequence (GEH . . . ).
  • the peptide GENT (SEQ ID NO. 15) is identical in family A and B, the junction between the 2 parts is the Gly residue.
  • Residue 155 of the full length sequence corresponds to the residue 136 of the mature sequence.
  • Recombinant plasmids ID Host strain Plate agar LVL489, LVL490, LVL491, LVL511, T7 LB + agar- LVL512, LVL513 and LVL514 Express A 1.5% B 40 ⁇ g/ml kanamycin c
  • B BBL TM Select APS TM LB broth base, BD, MD, USA (catalogue number: 292438); Agar, Laboratoire MAT, QC, Canada (catalogue number: AP-0108) C Sigma, ON, Canada (catalogue number: K-4000)
  • Cultures were incubated at 37° C., 250 RPM until an O.D. 600 nm around 0.8. At this time, 1 ml of each culture was collected, centrifuged at 14 000 RPM for 5 minutes and supernatants/pellets were frozen at ⁇ 20° C. separately.
  • O.D. 600 nm was evaluated after induction and culture was centrifuged at 14 000 RPM for 5 minutes and supernatant/pellets were frozen at ⁇ 20° C. separately.
  • Bacterial pellet was resuspended in 20 mM bicine buffer (pH 8.0) containing 500 mM NaCl and a mixture of protease inhibitor (Complete, Roche). Bacteria were lysed using a Constant System 1.1 KW 2 ⁇ 30 000 PSI. Soluble (supernatant) and insoluble (pellet) components were separated by centrifugation at 20 000 g for 20 min at 4° C.
  • the 6-His tagged-protein was purified under native conditions on IMAC using Profinia standard protocol (flow rate: 2 ml/min).
  • the soluble components were loaded on a 5 ml H is Trap column (BioRad) preequilibrated with the same buffer used to bacterial resuspension. After loading on the column, the column was washed with the same buffer.
  • 20 mM bicine buffer (pH 8.0) containing 500 mM NaCl, 10 mM imidazole for the second wash. Elution was performed using a 20 mM bicine buffer (pH 8.0) containing 500 mM NaCl and 250 mM imidazole. Proteins were dialysed using PBS 1 ⁇ pH 7.4 and dosing was determined using DC Protein Assay of BioRad.
  • Membranes were blocked for 30 minutes at 37° C., 60 RPM using 3% milk/PBS 1 ⁇ fresh solution. After the blocking incubation, Primary Antibodies were added consisting to ⁇ -6 ⁇ His Tag (AbCam, catalogue number: ab9108-100) or ⁇ -fHbpA (200802032 pool g2, 18/06/08 D42) at dilution: 1:1000 or 1:400 respectively in 3% milk/PBS 1 ⁇ fresh solution for 1 hour at 37° C., 60 RPM. After that, membranes were washed three times 5 minutes at room temperature using 0.02% Tween 20/PBS 1 ⁇ .
  • ⁇ -6 ⁇ His Tag AbCam, catalogue number: ab9108-100
  • ⁇ -fHbpA 200802032 pool g2, 18/06/08 D42
  • This fusion comprises a part of the family B MC58 mature protein sequence (from residue 1 to residue 135) and a part of the family A 8047 mature protein sequence (from residue 136 to residue 254).
  • 2 residues Glu217 and Thr238, identified by M. C. Schneider et al. as involved in the factor H-binding function of the protein, are mutated in Alanine.
  • the underlined sequence is the family B sequence coming from strain MC58.
  • the other part of the fusion is the family A sequence coming from strain 8047.
  • This fusion is based on fusion A in which only the amino acid Glu217 (conserved in all analysed strains and very probably involved in the factor H-binding) is mutated in Ala217.
  • This fusion based on fusion A, further including some mutations were introduced to restore the family B MAb502 epitope that is lost in fusions A and B.
  • the residue Gly147 is already present in the fHbp family A mature protein sequence.
  • the amino acids Asp146, Lys148 and Ser203 of fHbp family A protein sequence are replaced by Glu146, Arg149 and Arg204, respectively.
  • a Glycine is introduced at position 147.
  • Fusion protein D Is based on fusion C in which only the amino acid Glu218, involved in the factor H-binding, is mutated in Ala218.
  • This fusion is based in fusion protein C. Some additional residues were identified as potentially involved in the MAb502 recognition. There are Pro145, Phe227, Gly228, Lys230 and Glu233 in the family B mature protein sequence.
  • the fusion E is the fusion C in which these residues were inserted.
  • fHBPs variants (v) A and B were produced in BL21 (DE3) E. coli after IPTG induction and purification using IMAC column.
  • the fhbp sequences were derived from strains MC58 (fHBP B) and 2996 (fHBP A). Genes were cloned in pET24b plasmid without the nucleotide sequence corresponding to the leader sequence and with a His-Tag in C-ter.
  • mice were immunized three times by the intramuscular (IM) route on day 0, 21 and 28. Each injection contained 5 ⁇ g of monovalent fHBP vaccine (fHBP A or fHBP B) adsorbed onto Al(OH) 3 . On day 42, blood samples were taken for serum. Mice sera were from experiments 20080790 and 20090265.
  • IM intramuscular
  • fHBP A or fHBP B monovalent fHBP vaccine
  • guinea-pigs were immunized three times by the intramuscular (IM) route on day 0, 14 and 28. Each injection contained either OMV antigen normalized to 10 ⁇ g of protein and formulated with AIPO 4 . On day 42, blood samples were taken for serum. Guinea pig sera were from experiments 20090266.
  • IM intramuscular
  • N. meningitidis strains were cultivated overnight on Petri Dishes at 37° C. +5% CO 2 . They were sub-cultured for 4 hours on Petri Dishes without or with 20 ⁇ M TPEN (zinc chelator) 37° C. +5% CO 2 . Serum samples (pooled sera) were inactivated for 40 min at 56° C. and then diluted 1/10 or 1/50 in PBS-glucose 0.1% and then twofold diluted in a volume of 25 ⁇ l in flat bottom microplates.
  • Anti-fHbpB sera were tested in SBA with strain expressing the fHbp family B (M01-240101), while anti-fHbpA sera were used in SBA against strain expressing the fHbp from family A (M01-240013). SBA were performed in absence or presence of TPEN.
  • strain M01-240101 Only one strain was killed by anti-fHbp antibodies (strain M01-240101) in presence of complement. The second strain, M01-240013 is not killed by anti-fHbp antibodies. As observed, bactericidal culture condition (absence or presence of TPEN) has no impact on SBA titers.
  • Anti-fHbp antibodies were not able, alone, to provide effective killing of M01-240013 strain (from clonal complex ST269)
  • mice were immunized three times by the intramuscular (IM) route on day 0, 21 and 35. Each injection contained 5 ⁇ g of chimeric fHbp proteins adsorbed onto Al(OH) 3 . On day 49, blood samples were taken for serum. Mice sera were from experiment 20090833.
  • IM intramuscular
  • N. meningitidis strains were cultivated overnight on Petri Dishes at 37° C. +5% CO 2 . They were sub-cultured for 4 hours on Petri Dishes at 37° C. +5% CO 2 .
  • Serum samples (individual sera) were inactivated for 40 min at 56° C. and then diluted 1/50 in PBS-glucose 0.1% and then twofold diluted (8 ⁇ ) in a volume of 25 ⁇ l in flat bottom microplates. Then 25 ⁇ l of a mix of bacteria, from agar-plate culture (diluted in PBS-glucose 0.1% to yield ⁇ 50-250 CFU per well) and baby rabbit complement (final concentration in microwell: 12.5% v/v) was added to the serum dilution.
  • titer was set at 50 when killing for first dilution was below 50% killing or titer was set at 25600 if killing was higher than 50% at last dilution.
  • Sera were tested in rSBA with strain expressing the fHbp family B (H44/76) or the fHbp family A (S3446) (table 1 below).
  • Immunisation with recombinant fHbpB induced the production of bactericidal antibodies able to mediate the complement killing of H44/76 strain (fHbp family B) but not S3446 strain (fHbp family A) (percentage of responders 100% and 10%, respectively).
  • the recombinant fHbpA induced a low bactericidal antibody response againt the S3446 strain and a very low response against the H44/76 strain.
  • Anti-chimeric fHbp antibodies were able to provide effective killing of strains from both fHbp family (A and B). This ability is not altered by mutation of the fH binding site.
  • Map Native cleaved Map was purified from supernatant obtained after fermentation of H44/76 cps-strain. Two lots were produced, the second lot being obtained from H44/76 cps-overexpressing Map [achieved by amplifying the entire map gene from H44/76 by PCR and cloning in a Neisserial replicative plasmid derived from pFP10 (Pagotto et al, Plasmid 43, 24-34, 2000), containing a lacI Q gene and a tandem lac/tac promoter for controlled expression of Map]. Map was purified by concentration and chromatography steps.
  • Recombinant Map N-ter (aa 43-1178) was produced by cytoplasmic expression in E. coli.
  • fHbps variants (v) A and B were produced in BL21 (DE3) E. coli after IPTG induction and purification using IMAC column.
  • the fHbp sequences were derived from strains MC58 (fHbp B) and 2996 (fHbp A). Genes were cloned in pET24b plasmid without the nucleotide sequence corresponding to the leader sequence and with a His-Tag in C-ter.
  • mice were immunized three times by the intramuscular (IM) route on day 0, 14 and 28. Each injection contained 10 ⁇ g of native cleaved Hap or 5 ⁇ g of rec N-ter Hap formulated with specol. On day 42, blood samples were taken for serum. Mice sera were from experiments 20090608, 20100463, 20100708.
  • IM intramuscular
  • guinea-pigs were immunised three times by the intramuscular (IM) route on day 0, 14 and 28. Each injection contained 10 ⁇ g of protein formulated with specol. On day 42, blood samples were taken for serum. Guinea pig sera were from experiments 20090619, 20100464, 20100711.
  • IM intramuscular
  • mice were immunized three times by the intramuscular (IM) route on day 0, 21 and 28. Each injection contained 5 ⁇ g of monovalent fHbp vaccine (fHbp A or fHbp B) adsorbed onto Al(OH) 3 . On day 42, blood samples were taken for serum. Mice sera were from experiments 20080790 and 20090265.
  • IM intramuscular
  • fHbp A or fHbp B monovalent fHbp vaccine
  • N. meningitidis strains were cultivated overnight on Petri Dishes at 37° C. +5% CO 2 . They were sub-cultured for 4 hours on Petri Dishes with 20 ⁇ M TPEN (zinc chelator) at 37° C. +5% CO 2 . Serum samples (pooled sera) were inactivated for 40 min at 56° C. and then diluted 1/50 in PBS-glucose 0.1% and then twofold diluted in a volume of 25 ⁇ l in flat bottom microplates.
  • Strain 17540 was a gift from Julio Vasquez (CNM, Madrid, Spain), strain M01-240355 and NZ98/254 from R. Borrow (HPA, Manchester, UK).
  • the map/hap::kanR plasmid consisting in a kanamycine resistance cassette inserted into the unique PstI site of the H44/76 hap gene (van Ulsen et al, 2003) was a kind gift of Prof. Tommassen. Kanamycin-resistant colonies were screened for the inactivation of the hap gene by
  • Map induces a cross protective bactericidal response as strain M06-240336 with the lowest homology with H44/76 is killed. 17/22 strains are killed with anti-native cleaved Map antibodies obtained from guinea pigs. Six of these 17 strains are not killed by anti-fHbp antibodies. Difference of expression level of Map could be an explanation for absence of killing some strains as suggested by the facts that strains sharing similar Map sequences are killed (M06-240336) or not killed (M06-240877).
  • Map is the target of bactericidal antibodies.
  • ⁇ Map strains NZ98/254, M05-240355, SP17540 were used in bactericidal assays (table 2 below). In such SBA conditions, no killing was observed. These results confirm Map as a major antigen to induce cross bactericidal antibodies.
  • Map induces cross-bactericidal activity and provide effective killing of strains not killed by anti-fHbp including strains from clonal complex ST269 (eg M01-240013 strain) and strains from L2 immunotype (eg SP17540 strain).
  • Outer membranes vesicles were produced using classical 0.5% DOC extraction from recombinant H44/76 strain (porA KO, capsule minus, galE LOS and over-producing Msf and/or ZnuD protein).
  • fHbps variants (v) A and B were produced in BL21 (DE3) E. coli after IPTG induction and purification using IMAC column.
  • the fHbp sequences were derived from strains MC58 (fHbp B) and 2996 (fHbp A). Genes were cloned in pET24b plasmid without the nucleotide sequence corresponding to the leader sequence and with a His-Tag in C-ter.
  • mAb Hsfcross/5 was obtained from fusion of myeloma cells and lymphocyte B obtained from BALB/c mice immunized with 20 ⁇ g of OMVs from recombinant H44/76 strain (porA KO, capsule minus over-producing Msf from strain M01-0240101) using a repetitive, multiple site immunization strategy designated RIMMS.
  • GP guinea-pigs
  • fHbp A or fHbp B monovalent fHbp vaccine
  • N. meningitidis strains were cultivated overnight on Petri Dishes at 37° C. +5% CO 2 . They were sub-cultured for 4 hours on Petri Dishes at 37° C. +5% CO 2 . Serum samples (pooled sera) were inactivated for 40 min at 56° C. and then diluted 1/50 in PBS-glucose 0.1% and then twofold diluted in a volume of 25 ⁇ l in flat bottom microplates. Then 25 ⁇ l of a mix of bacteria, from agar-plate culture (diluted in PBS-glucose 0.1% to yield ⁇ 50-250 CFU per well) and baby rabbit complement (final concentration in microwell: 12.5% v/v) was added to the serum dilution.
  • N. meningitidis B strain 17567 (from J. Vasquez, CNM, Madrid, Spain) growth and transformation procedure were performed as described previously (Weynants et al, 2009).
  • the msf/hsf::CmR plasmid was constructed as followed.
  • a DNA fragment of 4771 bp corresponding to the 1531 bp 5′ flanking region of hsf gene, the 1775 bp of hsf coding sequence and the 1465 bp 3′ flanking region was PCR amplified from H44/76 genomic DNA with primers Hsf sens (CGCAATAAATGGGGTTGTCAATAATTGT) and Hsf reverse (AGTCAAGGCGCACGCTGTCGGCAT) and cloned in pGEMT-Easy vector.
  • the plasmid was then submitted to circle PCR mutagenesis with primers HSF5′ci2 (gaagatctgccgtctgaaacccgtaccgatgcggaaggctata) and HSF3′ci2 (gaagatctttcagacggcgataaagtcctgccgcgttgtgttttc) in order to (i) delete hsf gene, (ii) insert uptake sequences and (iii) insert BglII restriction sites allowing easy cloning of the antibiotic resistance gene.
  • the CmR gene was amplified from pCMC plasmid (Weynants et al, 2009) using primers BAD20 (tcccccgggagatctcactagtattaccctgttatccc) and CAM3′Bgl2 (agatctgccgctaactataacggtcc) primers. This fragment was cloned into the circle PCR product after BglII restriction resulting in plasmid pRIT15456. Chloramphenicol-resistant colonies were screened for the inactivation of the msf/hsf gene by PCR on boiled bacterial lysate. Absence of Msf expression was further confirmed by Western Blot. Integrity of LOS was also checked by Tricine gel and Silver staining for all clones to avoid changes in complement sensitivity.
  • strain SP17567 with a high level of Msf expression (western blotting results) was selected.
  • Results in table 1 below show an increase of at least 20 fold of bactericidal activity for Msf overexpressing blebs compared to control blebs. High bactericidal titer is also observed with Msf specific mAb. Only the recombinant fHbpA induces a low bactericidal response around the detection limit of the test.
  • Msf is the major target of bactericidal antibodies
  • ⁇ Msf SP17567 strains was used in bactericidal assay. In such SBA conditions, no killing was observed.
  • Anti-Msf antibodies could mediate the killing of a wild type strain that is poorly killed by anti-fHbp antibodies. This suggests that the addition of Msf to a fHbp based vaccine could enhance the coverage of a MenB vaccine.
  • Outer membranes vesicles were produced using classical 0.5% DOC extraction from recombinant H44/76 strain (porA KO, capsule minus, galE LOS and over-producing ZnuD (and Msf) protein).
  • mice were immunized three times by the intramuscular (IM) route on day 0, 21 and 28.
  • IM intramuscular
  • GP guinea-pigs
  • Each injection contained OMV antigen normalized to 5 (mice) or 10 ⁇ g (GP) of protein and formulated with AlPO 4 .
  • Mice and guinea pig sera were from experiments 20090265, 20100463 and 20090266, 20100464 respectively.
  • mice were immunized three times by the intramuscular (IM) route on day 0, 21 and 35. Each injection contained 5 ⁇ g of monovalent fHbp vaccine (fHbp A or fHbp B) adsorbed onto Al(OH) 3 . On day 49, blood samples were taken for serum. Mice sera were from experiment 20090833.
  • IM intramuscular
  • N. meningitidis strains were cultivated overnight on Petri Dishes at 37° C. +5% CO 2 . They were sub-cultured for 4 hours on Petri Dishes without or with 20 ⁇ M TPEN (zinc chelator) 37° C. +5% CO 2 . Serum samples (pooled sera) were inactivated for 40 min at 56° C. and then diluted 1/50 in PBS-glucose 0.1% and then twofold diluted in a volume of 25 ⁇ l in flat bottom microplates.
  • N. meningitidis strains growth and transformation procedure were performed as described previously (Weynants et al, 2009). When needed, induction of ZnuD expression was obtained by adding 20 mM TPEN (N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine) in the medium. Strain 17540 was a gift from Julio Vasquez (CNM, Madrid, Spain).
  • the znuD::kanR plasmid was a kind gift of Prof. Tommassen and is described in Stork et al, 2010. Kanamycin-resistant colonies were screened for the partial deletion of the znuD gene by PCR on boiled bacterial lysate. ZnuD inactivation was further confirmed by Western blot on whole cell lysate after growth in presence of TPEN. Integrity of LOS was checked by Tricine gel and Silver staining for all clones to avoid changes in complement susceptibility.
  • the evaluation of the bactericidal potential of ZnuD antibodies was performed via the use of zinc-restricted growth media (like in-vivo conditions). This was achieved by using 20 ⁇ M of TPEN in MH agar plates.

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