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US20060057160A1 - Vaccine composition - Google Patents

Vaccine composition Download PDF

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US20060057160A1
US20060057160A1 US10/523,055 US52305505A US2006057160A1 US 20060057160 A1 US20060057160 A1 US 20060057160A1 US 52305505 A US52305505 A US 52305505A US 2006057160 A1 US2006057160 A1 US 2006057160A1
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Prior art keywords
los
gene
strain
blebs
neisserial
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Inventor
Ralph Biemans
Philippe Denoel
Christiane Feron
Carine Goraj
Michael Jennings
Jan Poolman
Vincent Weynants
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University of Queensland UQ
GlaxoSmithKline Biologicals SA
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Priority claimed from GB0218037A external-priority patent/GB0218037D0/en
Priority claimed from GB0218051A external-priority patent/GB0218051D0/en
Priority claimed from GB0218035A external-priority patent/GB0218035D0/en
Priority claimed from GB0218036A external-priority patent/GB0218036D0/en
Priority claimed from GBGB0220197.8A external-priority patent/GB0220197D0/en
Priority claimed from GBGB0220199.4A external-priority patent/GB0220199D0/en
Priority claimed from GB0225524A external-priority patent/GB0225524D0/en
Priority claimed from GB0225531A external-priority patent/GB0225531D0/en
Priority claimed from GB0230168A external-priority patent/GB0230168D0/en
Priority claimed from GB0230170A external-priority patent/GB0230170D0/en
Priority claimed from GB0230164A external-priority patent/GB0230164D0/en
Priority claimed from GB0305028A external-priority patent/GB0305028D0/en
Application filed by Individual filed Critical Individual
Assigned to GLAXOSMITHLKINE BIOLOGICALS S.A., UNIVERSITY OF QUEENSLAND OF BRISBANE, THE reassignment GLAXOSMITHLKINE BIOLOGICALS S.A. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: JENNINGS, MICHAEL PAUL, PERON, CHRISTIANE, BIEMANS, RALPH, DENOEL, PHILIPPE, GORAJ, CARINE, POOLMAN, JAN, WEYNANTS, VINCENT
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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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • 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
    • A61K39/102—Pasteurellales, e.g. Actinobacillus, Pasteurella; Haemophilus
    • 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
    • A61K39/104—Pseudomonadales, e.g. Pseudomonas
    • A61K39/1045—Moraxella
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • 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
    • 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
    • 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
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/521—Bacterial cells; Fungal cells; Protozoal cells inactivated (killed)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of neisserial vaccine compositions, their manufacture, and the use of such compositions in medicine. More particularly it relates to processes of making novel engineered meningococcal strains which are less phase variable in terms of their LOS immunotype, and from which novel LOS subunit or meningococcal outer-membrane vesicle (or bleb) vaccines can be derived.
  • Neisseria meningitidis is a Gram negative bacterium frequently isolated from the human upper respiratory tract. It is a cause of serious invasive bacterial diseases such as bacteremia and meningitis. The incidence of meningococcal disease shows geographical, seasonal and annual differences (Schwartz, B., Moore, P. S., Broome, C. V.; Clin. Microbiol. Rev. 2 (Supplement), S18-S24, 1989). The bacterium is commonly classified according to the serogroup if its capsular polysaccharide.
  • Neisseria meningitidis infections has risen in the past few decades in many European countries. This has been attributed to increased transmission due to an increase in social activities (for instance swimming pools, theatres, etc.). It is no longer uncommon to isolate Neisseria meningitidis strains that are less sensitive or resistant to some of the standard antibiotics. This phenomenon has created an unmet medical need and demand for new anti-microbial agents, vaccines, drug screening methods, and diagnostic tests for this organism.
  • a serogroup B vaccine is not available.
  • the serogroup B capsular polysaccharide has been found to be nonimmunogenic—most likely because it shares structural similarity with host components (Wyle, F. A., Artenstein, M. S., Brandt, M. L. et al. J. Infect. Dis. 126: 514-522, 1972; Finne, J. M., Leinonen, M., Gurkelä, P. M. Lancet ii.: 355-357, 1983). Effort has therefore been focused in trying to develop serogroup B vaccines from outer membrane vesicles (or blebs) or purified protein components therefrom.
  • meningococcal lipooligosaccharides are outer membrane bound glycolipids which differ from the lipopolysaccharides (LPS) of the Enterobacteriaceae by lacking the O side chains, and thus resemble the rough form of LPS (Griffiss et al. Rev Infect Dis 1988; 10: S287-295). Heterogeneity within the oligosaccharide moiety of the LOS generates structural and antigenic diversity among different meningococcal strains (Griffiss et al. Inf. Immun. 1987; 55: 1792-1800). This has been used to subdivide the strains into 12 immunotypes (Scholtan et al.
  • Immunotypes L3, L7, & L9 are immunologically identical and are structurally similar (or even the same) and have therefore been designated L3, 7, 9 (or, for the purposes of this specification, generically as “L3”).
  • Meningococcal LOS L3, 7, 9 (L3), L2 and L5 can be modified by sialylation, or by the addition of cytidine 5′-monophosphate-N-acetylneuraminic acid.
  • L2, L4 and L6 LOS are distinguishable immunologically, they are structurally similar and where L2 is mentioned herein, either L4 or L6 may be optionally substituted within the scope of the invention.
  • Antibodies to LOS have been shown to protect in experimental rats against infection and to contribute to the bactericidal activity in children infected with N. meningitidis (Griffiss et al J Infect Dis 1984; 150: 71-79).
  • LOS is also present on the surface of meningococcal blebs.
  • meningococcal outer membrane vesicle (or bleb) based vaccines de Moraes, J. C., Perkins, B., Camargo, M. C. et al. Lancet 340: 1074-1078, 1992; Bjune, G., Hoiby, E. A. Gronnesby, J. K. et al. 338: 1093-1096, 1991.
  • Such vaccines have the advantage of including several integral outer-membrane proteins in a properly folded conformation which can elicit a protective immunological response when administered to a host.
  • Neisserial strains including N.
  • meningitidis serogroup B—menB excrete outer membrane blebs in sufficient quantities to allow their manufacture on an industrial scale. More often, however, blebs are prepared by methods comprising a 0.5% detergent (e.g. deoxycholate) extraction of the bacterial cells (e.g. EP 11243). Although this is desired due to the toxicity of LOS (also called endotoxin) as described above, it also has the effect removing most of the LOS antigen from the vaccine.
  • LOS also called endotoxin
  • LOS also known as LPS or lipopolysaccharide
  • LPS lipopolysaccharide
  • a further problem with using LOS as a vaccine antigen is that 12 LPS immunotypes exist with a diverse range of carbohydrate-structures (M. P. Jennings et al, Microbiology 1999, 145, 3013-3021; Mol Microbiol 2002, 43:931-43). Antibodies raised against one immunotype fail to recognise a different immunotype. Although effort has been focused on producing a generic “core” region of the oligosaccharide portions of the LOS immunotypes (e.g. WO 94/08021), the bactericidal activity of antibodies generated against the modified LOS is lost. Thus a vaccine may need to have many LOS components of different immunotype to be effective.
  • meningococcal LOS or blebs containing LOS
  • a feature of meningococcal LOS is the reversible, high frequency switching of expression (phase variation) of terminal LOS structures (M. P. Jennings et al, Microbiology 1999, 145, 3013-3021).
  • the phase variation exhibited by the LOS is an obstacle to the development of a cross-protective OMV or subunit vaccine based on the use of LOS as a protective antigen.
  • MenB strain H44/76 for example, the rate of switching from L3 to L2 immunotype is estimated at 1 in 1000 to 5000. Antibodies raised against the L3 structure failed to recognize the L2 immunotype and vice versa. Therefore it is extremely hard to maintain a LOS or bleb production strain with a constant, homogenous LOS immunotype.
  • the present invention presents processes for ameliorating one or more of the above problems, and presents methods for making novel vaccines based on meningococcal LOS as a protective antigen, particularly when present on an outer membrane vesicle.
  • the present invention relates to processes of making vaccine compositions for the effective prevention or treatment of neisserial, preferably meningococcal, disease.
  • the processes of the invention involve making a genetically engineered meningococcal strain which has a fixed or locked LOS immunotype.
  • methods are disclosed which allow L2 and L3 LOS immunotypes to be fixed.
  • a process for making LOS or blebs from such engineered strains is further covered, as is a method of making an immunogenic composition comprising the steps of making the above LOS or blebs and mixing with a pharmaceutically acceptable excipient.
  • lipooligosaccharide (or “LOS”) may also be referred to as “lipopolysaccharide” or “LPS”.
  • a locus containing various lgt genes is required for the biosynthesis of the terminal LOS structure (the sequences of which are known in the art—see M. P. Jennings et al, Microbiology 1999, 145, 3013-3021 and references cited therein; J. Exp. Med. 180:2181-2190 [1994]; WO 96/10086).
  • Meningococci can change the immunotype of the expressed LOS via a mechanism of phase variable expression of some of these genes.
  • the phase variable expression of LOS in L3 type menB strains e.g. MC58, H44/76) operates via high frequency mutations in a homopolymeric G tract region of lgtA.
  • L2 and L3 (and other) immunotypes A major difference between L2 and L3 (and other) immunotypes is the presence or absence of a glucose residue on the second heptose (see FIG. 1 [with grey arrows showing phase variation] and FIG. 2 ). The addition of this residue is catalyzed by the lgtG gene product, which also exhibits phase variable expression.
  • Other strains e.g. 126E
  • the present inventors have overcome this problem by developing methods of producing neisserial vaccine production strains which are fixed (i.e. not phase variable) in their LOS immunotype.
  • a genetically engineered neisserial (preferably meningococcal, most preferably serogroup B) strain comprising the step of genetically engineering a neisserial (preferably meningococcal) strain with phase-variable LOS synthesis, to render LOS expression less phase variable (and preferably non-phase variable or fixed).
  • phase variability or “less phase variable” in terms of LOS immunotype it is meant that one or more (preferably all) phase variable genes involved in the synthesis of the LOS immunotype or related LOS immunotypes is made less phase-variable or fixed so that the rate of switching between immunotypes is reduced (preferably by more than 2, 3, 5, 10 or 50 fold).
  • fixed and non-phase variable in terms of LOS immunotype it is meant that one or more (preferably all) phase variable genes involved in the synthesis of the LOS immunotype or related LOS immunotypes is fixed or made non-phase variable.
  • reduced phase variability or “less phase variable” in terms of LOS biosynthesis gene expression, it is meant that the chance of switching functional gene expression between on and off is reduced (preferably by more than 2, 3, 5, 10 or 50 fold), and “fixed” and “non-phase variable” in terms of LOS biosynthesis gene expression means that a gene previously susceptible to phase variation is rendered not susceptible to phase variation beyond the background chance of non site-specific switching on or off of functional gene expression.
  • the process results in a reduced (preferably non) phase variable LOS having, preferably exclusively, an L2 immunotype (most preferably constitutively synthesised).
  • an L2 strain is used to perform this process of the invention.
  • such a process has a genetic engineering step comprising the element of reducing phase-variability of (preferably fixing) expression of the lgtA and/or lgtG gene products (i.e. fixing such that expression of full-length, functional gene product may not be switched off by phase variation—either or both the genes are constitutively expressed).
  • the expression of either or both of lgtA and lgtG gene products is reduced in phase variability (preferably fixed) by reducing the length of the homopolymeric nucleotide tract (see Jennings et al. Microbiology 1999 145:3013) within the open-reading frame of the respective gene whilst maintaining the open-reading frame of the gene in frame.
  • the tract is reduced to 8, more preferably 2, or most preferably 5 consecutive G nucleotides.
  • the gene with 5 consecutive G nucleotides was optimal in terms of reduction of tract length and maintenance of LgtA enzyme function.
  • a preferred embodiment is therefore a reduction of the tract to 5 nucleotides in combination with altering the codon usage within the tract as described below.
  • the tract is reduced to 8, 5 or 2 consecutive C nucleotides.
  • tract reductions can be simply performed in general using homologous recombination (see WO 01/09350) between a plasmid construct containing the reduced tract and the genomic DNA of the strain to be changed after transformation of the strain with the plasmid.
  • the expression of lgtA gene product can be reduced in phase-variability (preferably fixed) by changing the sequence of the homopolymeric nucleotide tract within the open-reading frame of the lgtA gene such that: one or more GGG codons encoding Glycine is changed to any other codon encoding glycine (GGA, GGC or GGT), or a codon encoding a conservative mutation, and/or the TCG codon encoding Serine (the final G being part of the tract) is changed to any other codon encoding Serine, or a codon encoding a conservative mutation, whilst maintaining the open-reading frame of the gene in frame.
  • a 5G homopolymeric tract can advantageously have one GGG Glycine codon mutated to a nucleotide sequence GGG(A/C/T)G.
  • the expression of lgtG gene product can be alternatively or additively reduced in phase-variation (preferably fixed) by changing the sequence of the homopolymeric nucleotide tract within the open-reading frame of the lgtG gene such that: one or more CCC codons encoding Proline is changed to any other codon encoding Proline (CCA, CCG or CCT), or a codon encoding a conservative mutation, and/or the GCC codon encoding Alanine (the final CC pair being part of the tract) is changed to any other codon encoding Alanine, or a codon encoding a conservative mutation, whilst maintaining the open-reading frame of the gene in frame.
  • codons are replaced with codons encoding the same amino acids, however where conservative mutations are used it is preferred that: 1) codons are selected containing 2 or (preferably) fewer nucleotides of the type making up the tract, & 2) the new encoded amino acid is a conservative mutation. Conservation mutations are understood by skilled persons in this field. However preferred substitutions are detailed in the table below. Exemplary Preferred Original residue substitutions substitution A V, L, I V G P, A A P A, G A S T, A T
  • a combination of the above methods of the invention could be used to fix the lgtA and/or lgtG genes. For instance by both reducing the lgtA tract to 5 G residues, and replacing one of the GGG codons encoding Glycine to one of the other 3 codons encoding Glycine [yielding a final tract nucleotide sequence of GGG(A/C/T)G].
  • the expression of the lgtA gene product is fixed by reducing the length of the homopolymeric G nucleotide tract within the open-reading frame of the respective gene to 2 or 5 consecutive G nucleotides whilst maintaining the open-reading frame of the gene in frame, and the expression of lgtG gene product is fixed by changing the sequence of the homopolymeric nucleotide tract within the open-reading frame of the lgtG gene such that: 1, 2 or preferably 3 CCC codons encoding Proline is changed to any other codon encoding Proline (CCA, CCG, or CCT), or a codon encoding a conservative mutation, and/or the GCC codon encoding Alanine is changed to any other codon encoding Alanine, or a codon encoding a conservative mutation, whilst maintaining the open-reading frame of the gene in frame.
  • the process of the invention results in a reduced (preferably non) phase variable LOS having (preferably exclusively) an L3 immunotype (most preferably constitutively synthesised).
  • a reduced (preferably non) phase variable LOS having (preferably exclusively) an L3 immunotype (most preferably constitutively synthesised).
  • the genetic engineering step preferably comprises the elements of reducing phase-variability of (preferably fixing) the expression of the lgtA gene product [preferably such that expression of full-length, functional product may not be switched off by phase variation (i.e. is constitutively expressed as described above)], and/or permanently downregulating the expression of functional gene product from the lgtG gene.
  • downregulating the expression of functional gene product it is meant that additions, deletions or substitutions are made to the promoter or open reading frame of the gene such that the biosynthetic activity of the total gene product reduces (by 60, 70, 80, 90, 95 or most preferably 100%).
  • frameshift mutations may be introduced, or weaker promoters substituted, however most preferably most or all of the open reading frame and/or promoter is deleted to ensure a permanent downregulation of the gene product. See WO 01/09350 for further methods of gene downregulation.
  • the expression of lgtA gene product can be made less phase variable (preferably fixed) by reducing the length of the homopolymeric nucleotide tract within the open-reading frame of the gene whilst maintaining the open-reading frame of the gene in frame (preferably the homopolymeric G tract in the lgtA open-reading frame is reduced to 8, more preferably 2 or, most preferably, 5 consecutive G nucleotides) and/or by changing the sequence of the homopolymeric nucleotide tract within the open-reading frame of the lgtA gene such that: one or more GGG codons encoding Glycine is changed to any other codon encoding glycine, or a codon encoding a conservative mutation, and/or the TCG codon encoding Serine is changed to any other codon encoding Serine, or a codon encoding a conservative mutation, whilst maintaining the open-reading frame of the gene in frame (as described above).
  • a 5G homopolymeric tract can advantageously
  • a combination of the above methods of the invention could be used to fix the lgtA gene. For instance by both reducing the lgtA tract to 5 G residues, and replacing one of the GGG codons encoding Glycine to one of the other 3 codons encoding Glycine [yielding a final tract nucleotide sequence of GGG(A/C/T)G].
  • the expression of functional gene product from the IgtG gene is switched off (i.e. there is no or negligible IgtG gene product biosynthetic activity post mutation).
  • the expression of the lgtA gene product is fixed by reducing the length of the homopolymeric G nucleotide tract within the open-reading frame of the respective gene to 5 or 2 consecutive G nucleotides whilst maintaining the open-reading frame of the gene in frame, and the expression of functional gene product from the lgtG gene is switched off by deleting all or part of the promoter and/or open-reading frame of the gene.
  • the meningococcal strain to be altered has an lgtC gene (e.g. strain 126E)
  • the processes of the invention have a genetic engineering step which comprises an element of permanently downregulating the expression of functional gene product from the lgtC gene, preferably by switching the gene off, most preferably by deleting all or part of the promoter and/or open-reading frame of the gene.
  • inactivation of the lgtB gene results in an intermediate LOS structure in which the terminal galactose residue and the sialic acid are absent (see FIG. 1-3 , the mutation leaves a 4GlcNAc ⁇ 1-3Gal ⁇ 1-4Glc ⁇ 1-structure in L2 and L3 LOS).
  • Such intermediates could be obtained in an fixed L3 (lgtA fixed on and/or lgtG fixed off) and a fixed L2 (lgtA and/or lgtG fixed on) LOS strain.
  • An alternative and less preferred (short) version of the LOS can be obtained by turning off the lgtE gene.
  • the above processes may also have a genetic engineering step comprising the element of downregulating (preferably permanently) the expression of functional gene product from the lgtB or lgtE gene, preferably by switching the gene off, most preferably by deleting all or part of the promoter and/or open-reading frame of the gene.
  • a process involving rendering a strain lgtB ⁇ is most preferred as the inventors have found that this is the optimal truncation for resolving the safety issue whilst still retaining an L2 or L3 LOS protective oligosaccharide epitope that can still induce a bactericidal (and even cross-bactericidal) antibody response.
  • the strains used in the processes of the invention are unable to synthesise capsular polysaccharide, where this is not the case it is advantageous to add a further process step of downregulating the expression of functional gene product critical for the production of capsular polysaccharide.
  • the genetic engineering step of the process comprises the element of permanently downregulating the expression of functional gene product from the siaD gene, preferably by switching the gene off, most preferably by deleting all or part of the promoter and/or open-reading frame of the gene.
  • Such an inactivation is also 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.
  • the processes of the invention utilise a meningococcus B mutant strain with the lgtB and siaD genes downregulated or inactivated (preferably a lgtB ⁇ siaD ⁇ strain).
  • the 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, preferably by switching the gene off, most preferably by deleting all or part of the promoter and/or open-reading frame of the gene.
  • the lgtE ⁇ mutation may be combined with one or more of these mutations.
  • the lgtB ⁇ mutation is combined with one or more of these mutations.
  • a Neisserial locus containing various lgt genes, including lgtB and lgtE, and its sequence is known in the art (see M. P. Jennings et al, Microbiology 1999, 145, 3013-3021 and references cited therein; J. Exp. Med. 180:2181-2190 [1994]; WO 96/10086).
  • the processes of the invention may also include steps which render the LOS less toxic. Although this is not necessary for intranasal immunization with native OMV (J. J. Drabick et al, Vaccine (2000), 18, 160-172), for parenteral vaccination detoxification would present an advantage.
  • LOS can be detoxified genetically by mutation/modification/inactivation of genes involved in Lipid A biosynthesis for example by downregulating the expression of functional gene product from the msbB and/or htrB genes, preferably by switching the gene off, most preferably by deleting all or part of the promoter and/or open-reading frame of the gene.
  • one or more of the following genes may be upregulated (by introducing a stronger promoter or integrating an extra copy of the gene): pmrA, pmrB, pmrE and pmrF.
  • msbB and htrB genes of Neisseria are also called lpxL1 and lpxL2, respectively, (see WO 00/26384) and deletion mutations of these genes are characterised phenotypically by the msbB ⁇ mutant LOS losing one secondary acyl chain compared to wild-type (and retaining 4 primary and 1 secondary acyl chain), and the htrB ⁇ mutant LOS losing both secondary acyl chains.
  • Such mutations are preferably combined with mutations to ensure that the neisserial production strain is capsular polysaccharide deficient (see above) to ensure the optimal presentation of detoxified LOS on the bleb, or to aid the purification of the detoxified subunit LOS.
  • a further aspect of the invention is a process of isolating L2 LOS comprising the steps of producing a genetically engineered neisserial strain with a reduced phase variable (preferably fixed) L2 immunotype by the process of the invention as described above, and isolating L2 LOS from the resulting strain.
  • An additional advantageous step may be added to this process, namely conjugating the L2 LOS to a carrier comprising a source of T-cell epitopes (rendering the LOS an even better immunogen) and/or the step of presenting the L2 LOS in liposome formulations known in the art (see for instance WO 96/40063 and references cited therein).
  • a carrier comprising a source of T-cell epitopes is usually a peptide or, preferably, a polypeptide or protein. Conjugation techniques are well known in the art. Typical carriers include protein D from non typeable H. influenzae , tetanus toxoid, diphtheria toxoid, CRM197, or outer membrane proteins present in bleb (particularly neisserial or meningococcal) preparations. Preferably the oligosaccharide portion of the LOS is conjugated.
  • a still further aspect of the invention is a process of isolating L3 LOS comprising the steps of producing a genetically engineered meningococcal strain with a reduced phase variable (preferably fixed) L3 immunotype by the process of the invention as described above, and isolating L3 LOS from the resulting strain.
  • An additional advantageous step may be added to this process, namely conjugating the L3 LOS to a carrier comprising a source of T-cell epitopes and/or the step of presenting the L3 LOS in a liposome formulation.
  • the LOS is detoxified as part of the process. This may be done by known techniques of hydrazine or alkaline hydrolysis chemical treatments which remove acyl chains from the molecule (but which may reduce the protective efficacy of the molecule), but is preferably done by isolating the LOS from an htrB ⁇ and/or msbB ⁇ meningococcal mutant (as described above; particularly in capsule polysaccharide minus strains), or by adding a non-toxic peptide functional equivalent of polymyxin B [a molecule with high affinity to Lipid A] to the isolated LOS, in particular SAEP 2.
  • outer membrane proteins may optionally also be added, and the LOS may be conjugated intra-liposome to such outer membrane proteins to render the oligosaccharide a T-dependent antigen. This may be done with a similar chemistry as described for intra-bleb LOS cross-linking as described below.
  • a further aspect of the invention is a process of isolating meningococcal blebs having an L2 or L3 LOS immunotype, comprising the steps of producing a genetically engineered meningococcal strain with a reduced phase variable (preferably fixed) L2 or L3 immunotype, respectively, by the processes of the invention as described above; and isolating blebs from the resulting strain.
  • Outer Membrane Vesicles can be isolated by many known techniques (Fredriksen et al, NIPH Annals (1991), 14, 67-79; Zollinger et al, J. Clin Invest (1979), 63, 836-848; Saunders et al, Infect Immun (1999), 67, 113-119; J. J. Drabick et al, Vaccine (1999), 18, 160-172). These divide into 2 main groups—techniques which use deoxycholate (about 0.5%) to extract blebs from meningococcus, and techniques that use low levels of deoxycholate (DOC) or no deoxycholate at all.
  • DOC deoxycholate
  • DOC free process blebs have the interesting feature of maintaining high level of LOS in the OMV—which is advantageous in a vaccine where LOS is a protective antigen.
  • concentration of L3 Ags in OMV obtained by a DOC free process is approximately ten times higher, also taking into account the fixing of lgtA.
  • a detergent-free (preferably DOC-free) process of preparing blebs is preferred for the purposes of the processes of this invention for this reason, although extraction with a buffer containing low levels of detergent (preferably DOC) may also be advantageous in that the step would leave most of the tightly interacting LOS in the bleb whilst removing any more toxic loosely retained LOS.
  • the LOS content of the blebs isolated by the process of the invention is 3-30, 5-25, 10-25, 15-22, and most preferably around or exactly 20% LOS content as measured by silver staining after SDS-PAGE electrophoresis using purified LOS as a standard (see method of Tsai, J. Biol. Standardization (1986) 14:25-33).
  • Nmen L3 LOS as a standard in this method, in general LOS content in Nmen L3 immunotype blebs extracted with 0.1% DOC is about 20% LOS, with 0.2% DOC is about 15% LOS, with 0.3% DOC is about 10% LOS, and with 0.5% DOC is about 5% LOS.
  • the above bleb isolation processes of the invention may comprise an additional advantageous step of conjugating the L2 or L3 LOS in situ to an outer membrane protein (e.g. PorA or PorB) also present in the bleb preparation.
  • an outer membrane protein e.g. PorA or PorB
  • a further aspect of the invention is a process of the invention where the isolated bleb preparation is conjugated (through an integral outer-membrane protein) to LOS.
  • LOS may be added to a bleb preparation for conjugation, it is preferred that the LOS is naturally present on the surface of the bleb preparation.
  • 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 the outer membrane.
  • conjugation of the LOS within the bleb can result in a detoxification of the LOS (without wishing to be bound by theory, the Lipid A portion may be more stably buried in the outer membrane if conjugated thus being less available to cause toxicity).
  • the processes of the invention may thus yield conjugated bleb preparations which are typically such that the toxicity of the LOS in the bleb is reduced compared to the same blebs with the same amount of totally unconjugated LOS.
  • LOS toxicity may be readily determined by a skilled person, for example using the LOS rabbit pyrogenicity assay in the European Pharmacopoeia.
  • a process of the invention yielding a composition 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 is advantageous in being part of a process to make a vaccine for the treatment or prevention of neisserial (preferably meningococcal) disease, wherein the process allows the vaccine to be of reduced toxicity and/or capable of inducing a T-dependent bactericidal response against LOS in its native environment.
  • This invention therefore further provides a method to make such an intra-bleb LOS conjugated bleb preparation from a strain of reduced phase variability (preferably fixed) LOS immunotype.
  • intra bleb it is meant that LOS naturally present in the bleb is conjugated to outer membrane protein present on the same bleb.
  • Such bleb preparations may be made by isolated blebs and then subjected them to known conjugation chemistries to link groups (e.g. NH 2 or COOH) on the oligosaccharide portion of LOS to groups (e.g. NH 2 or COOH) on bleb outer membrane proteins.
  • link groups e.g. NH 2 or COOH
  • cross-linking techniques using glutaraldehyde, formaldehyde, or glutaraldehyde/formaldehyde mixes may be used, but it is preferred that more selective chemistries are used such as EDAC or EDAC/NHS (J. V. Staros, R. W. Wright and D. M. Swingle. Enhancement by N-hydroxysuccinimide of water-soluble carbodiimide-mediated coupling reactions.
  • 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), or may be purified from most (more than 60, 70, 80, 90, or 99% removed) and preferably all contaminating capsular polysaccharide. In this way, the intra-bleb LOS conjugation reaction is much more efficient.
  • the blebs of the invention have been prepared such that the LOS content of the blebs is 3-30, 5-25, 10-25, 15-22, and most preferably around or exactly 20% LOS content as measured by silver staining after SDS-PAGE electrophoresis using purified LOS as a standard (see method of Tsai, J. Biol. Standardization (1986) 14:25-33). 20% LOS in meningococcal blebs can be achieved with a 0.1% low DOC extraction, which may remove losely held LOS molecules, but conserve the majority of the antigen.
  • the intra-bleb conjugated blebs made by the process of the invention are derived from meningococcus
  • the strain from which they are derived is a mutant strain that cannot produce capsular polysaccharide (e.g. one of the mutant strains described above, in particular siaD ⁇ ).
  • a typical L3 meningococcal strain that can be used for the present invention is the H44/76 menB strain.
  • a typical L2 strain is the B16B6 menB strain or the 39E meningococcus type C strain or strain 760676.
  • the process of the invention allows the detoxification of blebs to some 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 by 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] (preferably SEAP 2) to the bleb composition (as described above).
  • meningococcal blebs and immunogenic compositions comprising blebs can be made by the processes of the invention which have as an important antigen LOS of a certain immunotype (preferably L2 or L3) which is reproducibly made without phase variation, is reduced in toxicity (and preferably substantially non-toxic), devoid of autoimmunity problems, has a T-dependent character, and is present in its natural environment.
  • a certain immunotype preferably L2 or L3
  • Men A, C, Y or W capsular polysaccharides or oligosaccharides may also be conjugated onto an outermembrane protein of the bleb in a process of the invention as well. Although this could be done in the same reaction as LOS cross-linking, it is preferred that this is done in a separate (preferably later) reaction.
  • Intrableb conjugation should preferably incorporate 1, 2 or all 3 of the following process steps: conjugation pH should be greater than pH 7.0, preferably greater than or equal to pH 7.5 (most preferably under pH 9); conditions of 1-5% preferably 2-4% most preferably around 3% sucrose should be maintained during the reaction; NaCl should be minimised in the conjugation reaction, preferably under 0.1M, 0.05M, 0.01M, 0.005M, 0.001M, and most preferably 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 preferred process for intra-bleb conjugation.
  • EDAC/NHS is preferred to formalydehyde 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.
  • EDAC/mg bleb by protein measured by Lowry
  • 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.
  • DOC deoxycholate
  • a preferred process of the invention is therefore a process for producing intra-bleb conjugated LOS (preferably meningococcal) comprising the steps of producing reduced phase variable LOS, isolating blebs, conjugating blebs in the presence of EDAC/NHS at a pH between pH 7.0 and pH 9.0 (preferably around pH 7.5), in 1-5% (preferably around 3%) sucrose, and optionally in conditions substantially devoid of NaCl (as described above), and isolating the conjugated blebs from the reaction mix.
  • LOS preferably meningococcal
  • 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).
  • a process for making immunogenic compositions or vaccines comprising the steps of producing isolated L2 LOS by the process of the invention as described above and/or producing isolated meningococcal blebs having an L2 LOS immunotype by the processes of the invention as described above, and formulating the L2 LOS and/or blebs with a pharmaceutically acceptable excipient.
  • a process for making immunogenic compositions or vaccines comprising the steps of producing isolated L3 LOS by the process of the invention as described above and/or producing isolated meningococcal blebs having an L3 LOS immunotype by the processes of the invention as described above, and formulating the L3 LOS and/or blebs with a pharmaceutically acceptable excipient.
  • An advantageous process of the invention is a process of making a multivalent immunogenic composition or vaccine comprising the steps of producing one or both of isolated L2 LOS or isolated meningococcal blebs having an L2 LOS immunotype by the processes of the invention as described above, and producing one or both of isolated L3 LOS or isolated meningococcal blebs having an L3 LOS immunotype by the processes of the invention as described above, and mixing said L2 and L3 vaccine components together along with a pharmaceutically acceptable excipient.
  • the process mixes isolated L2 and L3 LOS together which are made as described above (most preferably conjugated and in a liposome formulation). More preferably the process mixes L2 and L3 blebs together which are made as described above.
  • Such compositions are advantageous as approximately 70% of meningococcus B immunotypes observed in disease isolates have an L3 structure, and 30% are L2.
  • the invention therefore describes a process which can yield a universal meningococcus B vaccine.
  • the process of making immunogenic compositions or vaccines as described above may have an additional step of adding one or more (2, 3 or 4) meningococcal polysaccharides or oligosaccharides (either plain or conjugated to a carrier comprising T-cell epitopes) from serogroups A, C, Y or W to the composition.
  • at least C is added (most preferably conjugated), and more preferably A and C or Y and C (preferably all conjugated) and most preferably A, C, Y and W (preferably all conjugated).
  • Suitable adjuvants include an aluminium salt such as aluminum hydroxide gel (alum) or aluminium phosphate (preferably aluminum hydroxide), 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 (preferably aluminum hydroxide)
  • alum aluminium phosphate
  • iron or zinc or may be an insoluble suspension of acylated tyrosine, or acylated sugars, cationically or anionically derivatised polysaccharides, or polyphosphazenes.
  • Suitable Th1 adjuvant systems include, Monophosphoryl lipid A, particularly 3-de-O-acylated monophosphoryl lipid A (or other non-toxic derivatives of LPS), and a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL) [or non toxic LPS derivatives] together with an aluminium salt, preferably aluminium phosphate.
  • Monophosphoryl lipid A particularly 3-de-O-acylated monophosphoryl lipid A (or other non-toxic derivatives of LPS)
  • monophosphoryl lipid A preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL) [or non toxic LPS derivatives]
  • an aluminium salt preferably aluminium phosphate.
  • An enhanced system involves the combination of a monophosphoryl lipid A and a saponin derivative particularly the combination of QS21 [or other saponin] and 3D-MPL [or non toxic LPS derivative] as disclosed in WO 94/00153, or a less reactogenic composition where the QS21 [or saponin] 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 preferred formulation that may be added.
  • Other adjuvants that may be added comprise a saponin, more preferably QS21 and/or an oil in water emulsion and tocopherol.
  • Unmethylated CpG containing oligo nucleotides (WO 96/02555) may also be added
  • 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).
  • An immunoprotective dose of vaccines can be administered via the systemic or mucosal route. These administrations may include injection via the intramuscular, intraperitoneal, intradermal or subcutaneous routes; or via mucosal administration to the oral/alimentary, respiratory, genitourinary tracts.
  • bleb quantity in each vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccinees. 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 1-100 ⁇ g of each bleb, preferably 5-50 ⁇ g, and most typically in the range 5-25 ⁇ g.
  • Methods of making ghost preparations (empty cells with intact envelopes) from Gram-negative strains are well known in the art (see for example WO 92/01791).
  • Methods of killing whole cells to make inactivated cell preparations for use in vaccines are also well known.
  • the processes concerning blebs described throughout this document are therefore applicable to the processes concerning ghosts and killed whole cells for the purposes of this invention.
  • LOS phase variation is particularly problematic when trying to grow high cell densities of cells in fermentors.
  • meningococcal LOS immunotype to change (in particular to shorter oligosaccharide chain LOS immunotypes). This can happen for L2 and L3 strains, and in particular truncated (e.g. lgtB) strains.
  • lgtA should be reduced in phase-variability, preferably fixed.
  • an L2 or L3 meningococcal strain is grown (where preferably lgtA should be reduced in phase-variability, preferably fixed).
  • high cell density it is meant a cell density of OD 450 10-19, preferably 12-16, in iron non-limiting conditions, or 6-12, preferably 8-10, in iron limited conditions.
  • the process may be extended by adding steps of isolating the LOS from the culture at high cell density.
  • the LOS may then be conjugated to a carrier and/or introduced into a liposome as discussed above.
  • a bleb isolation step may alternately be added to obtain blebs from the culture of high cell density. This should be done ideally with a low detergent, preferably DOC, % process, typically 0-0.3%, preferably 0.05-70.2%, most preferably around or exactly 0.1% deoxycholate.
  • the bleb LOS may advantageously be intra-bleb cojugated to an outer membrane protein also present in the blebs as described above.
  • the process may be extended to producing an immunogenic composition by formulating the LOS produced above with a pharmaceutically acceptable excipient.
  • a process of making a multivalent immunogenic composition comprising the steps of producing isolated LOS or isolated blebs of a certain immunotype (preferably L2) by the above process, and producing isolated LOS or isolated blebs of a different immunotype (preferably L3) by the above process, and mixing these LOS components components together along with a pharmaceutically acceptable excipient.
  • Genes encoding glycosyltransferases in Neisseria meningitidis often contain simple tandem repeats (for example, homopolymeric tracts) which mediate phase variation (high frequency reversible on/off switching of gene expression (Jennings et al 1995, Mol Micro 18 724; Jennings et al 1999, Microbiology 145 3013).
  • the repeated sequences in these genes are present in the open reading frame and are transcribed and translated into protein. Phase variation may be eliminated by reducing (in frame) the homopolymeric tract.
  • the latter primer incorporated the change in the lgtA sequence from 14G to 2G.
  • the resulting PCR product was cloned into pT7Blue (Novogen), to create plasmid pT7lgtAG2.
  • pT7Blue Novogen
  • pT7lgtAG2 To reconstitute to complete lgtA gene so that the plasmid could be used to transform the new allele into Neisseria meningitidis , a BssHII fragment from plasmid p1B11 (Jennings et al 1995, supra) was cloned into the BssHII site of pT7lgtAG2 in the correct orientation.
  • Nucleotide sequence analysis confirmed the correct orientation of the gene and that the sequence segment was identical to the corresponding section of the wild-type lgtA gene (Genbank accession NMU25839) apart from the alteration of the homopolymeric tract from 14 to 2 G residues.
  • variants of the lgtAG2 primer mutations were made so that a series of similar plasmids were created that contained lgtA alleles with 3, 4, 5, 7 and 10 G residues in the homopolymeric tract region.
  • phase variation of the homopolymeric tract can also be fixed by altering the poly G regions so that the GGG codons are replaced with alternative glycine codons arranged so that the same amino acid sequence is encoded, but the nucleotide sequence does not have a repetitive nature and is unlikely to phase vary (see lgtG example below).
  • a combination of the 2 methods could also be used—for instance the homopolymeric tract could be cut to 5 G residues & a GGG codon replaced with an alternative glycine codon.
  • the plasmid pT7lgtAG2 was linearized and used to transform Neisseria meningitidis strain MC58 ⁇ 3 containing an lgtA::kan mutation (Jennings et al 1995, supra). Positive colonies were detected by mAb 4A8B2 in colony-immunoblot (Jennings et al 1999, supra).
  • LgtA positive phenotype (L3 immunotype structure) of the transformants was the result of the transfer of the lgtAG2 allele to the chromosome was confirmed by PCR of the relevant section of the lgtA gene using primers Lic31 ext and Lic16ext: 5′-CGA TGA TGC TGC GGT CTT TTT CCA T-3′, followed by nucleotide sequencing with the same set of primers.
  • the resulting strain 2G2 had the genotype: MC58 parent strain; siaD::ery lgtAG2).
  • Strain 2G2 was subsequently transformed with the a plasmid containing an lgtB::kan mutation (Jennings et al 1995, supra) to create strain 2G2ecoNI, this strain had the genotype: MC58 parent strain; siaD::ery lgtAG2 lgtB::kan
  • the MC58 derivative strain used is B:P1.7.16, opc-, siaD-.
  • This strain was genetically modified to express either L3 (strain 2G2 [modified to reduce the homopolymeric tract to only 2 G nucleotides], lgtA fixed on) or an intermediate epitope (strain 2G EcoN1b-1, lgtA fixed on as with 2G2 but lgtB additionally turned off) or an LPS in short version (strain C6, lgtE off).
  • L3 strain 2G2 [modified to reduce the homopolymeric tract to only 2 G nucleotides], lgtA fixed on
  • an intermediate epitope strain 2G EcoN1b-1, lgtA fixed on as with 2G2 but lgtB additionally turned off
  • an LPS in short version strain C6, lgtE off.
  • OMV were produced according either a DOC process or DOC free process.
  • mice (10 per group) were immunized three times by the intramuscular route on Day 0, 20 and 28. They received 1 or 10 ⁇ g (protein content) of blebs formulated on Al(OH)3. Blood samples were taken on day 28 (post II) and day 42 (post III).
  • Bactericidal assays were done on pooled sera and using homologous strains (MC58 and H44/76) and two heterologous strains (M97250687 and M9725078) with baby rabbit serum as source of exogenous complement.
  • L3 (2g2) or intermediate (2gecon1b-1) epitope induces cross-bactericidal antibodies, while blebs from truncated LPS strain (C6) induce lower level of cross-reacting antibodies. This was particularly illustrated when 1 ⁇ g of OMV was injected.
  • DOC free blebs may also advantageously retain some proteins losely interacting with the OMVs such as lipoproteins.
  • strain Neisseria meningitidis strain 35E (L2 immunotype typing strain) as a template primer pair Lg1: 5′-ATG AAG CTC AAA ATA GAC ATT G-3′ and Lg21: 5′-ATC TGC GGG CGG CGG CGC GAC TTG GAT-3′, and primer pair LGdell8: 5′-GAA TTC GGA TCC AAC TGA TTG TGG CGC ATT CC-3′ and Lg2UP: 5′-TGC CGT CTG AAG ACT TCA GAC GGC TTA TAC GGA TGC CAG CAT GTC-3′ (underlined sequence denotes a Neisseria meningitidis uptake sequence) were used to produce two PCR products.
  • Lg1 5′-ATG AAG CTC AAA ATA GAC ATT G-3′
  • Lg21 5′-ATC TGC GGG CGG CGG CGC GAC TTG GAT-3′
  • primer pair LGdell8 5′-GAA
  • the resulting strain ⁇ 3lgtA2GlgtG::kan-3, had the genotype: MC58 parent strain; siaD::ery lgtAG2 lgtG::kan.
  • This strain was then transformed with plasmid pL2+ and screened for colonies with an L2 phenotype and screen by colony-immuno blots (Mn 42F12.32).

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Applications Claiming Priority (25)

Application Number Priority Date Filing Date Title
GB0218037.0 2002-08-02
GB0218037A GB0218037D0 (en) 2002-08-02 2002-08-02 Vaccine composition
GB0218051A GB0218051D0 (en) 2002-08-02 2002-08-02 Vaccine composition
GB0218035A GB0218035D0 (en) 2002-08-02 2002-08-02 Vaccine composition
GB0218035.4 2002-08-02
GB0218051.1 2002-08-02
GB0218036A GB0218036D0 (en) 2002-08-02 2002-08-02 Vaccine
GB0218036.2 2002-08-02
GBGB0220199.4A GB0220199D0 (en) 2002-08-30 2002-08-30 Mutant protein and refolding method
GBGB0220197.8A GB0220197D0 (en) 2002-08-30 2002-08-30 Refolding method
GB0220199.4 2002-08-30
GB0220197.8 2002-08-30
GB0225524A GB0225524D0 (en) 2002-11-01 2002-11-01 Vaccine composition
GB0225531.3 2002-11-01
GB0225531A GB0225531D0 (en) 2002-11-01 2002-11-01 Vaccine
GB0225524.8 2002-11-01
GB0230168.7 2002-12-24
GB0230170.3 2002-12-24
GB0230168A GB0230168D0 (en) 2002-12-24 2002-12-24 Vaccine composition
GB0230164.6 2002-12-24
GB0230170A GB0230170D0 (en) 2002-12-24 2002-12-24 Vaccine
GB0230164A GB0230164D0 (en) 2002-12-24 2002-12-24 Vaccine composition
GB0305028.3 2003-03-05
GB0305028A GB0305028D0 (en) 2003-03-05 2003-03-05 Vaccine
PCT/EP2003/008569 WO2004015099A2 (fr) 2002-08-02 2003-07-31 Compositions de vaccins

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US10/523,055 Abandoned US20060057160A1 (en) 2002-08-02 2003-07-31 Vaccine composition
US10/523,114 Abandoned US20060034854A1 (en) 2002-08-02 2003-07-31 Vaccine composition comprising transferrin binding protein and hsf from gram negative bacteria
US10/523,117 Abandoned US20060240045A1 (en) 2002-08-02 2003-07-31 Neisserial vaccine compositions comprising a combination of antigens
US10/523,044 Expired - Fee Related US7838014B2 (en) 2002-08-02 2003-07-31 Vaccine compositions comprising L2 and/or L3 immunotype lipooligosaccharides from LGTB- Neisseria meningitidis
US12/909,621 Expired - Fee Related US8221770B2 (en) 2002-08-02 2010-10-21 Vaccine compositions comprising L2 and/or L3 immunotype lipooligosaccharides from LGTB-neisseria meningitidis
US13/248,693 Abandoned US20120027800A1 (en) 2002-08-02 2011-09-29 Neisserial vaccine compositions comprising a combination of antigens
US13/299,007 Abandoned US20120064119A1 (en) 2002-08-02 2011-11-17 Neisserial vaccine compositions comprising a combination of antigens
US13/299,041 Abandoned US20120064120A1 (en) 2002-08-02 2011-11-17 Neisserial vaccine compositions comprising a combination of antigens
US14/844,989 Abandoned US20160082097A1 (en) 2002-08-02 2015-09-03 Neisserial vaccine compositions comprising a combination of antigens

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US10/523,114 Abandoned US20060034854A1 (en) 2002-08-02 2003-07-31 Vaccine composition comprising transferrin binding protein and hsf from gram negative bacteria
US10/523,117 Abandoned US20060240045A1 (en) 2002-08-02 2003-07-31 Neisserial vaccine compositions comprising a combination of antigens
US10/523,044 Expired - Fee Related US7838014B2 (en) 2002-08-02 2003-07-31 Vaccine compositions comprising L2 and/or L3 immunotype lipooligosaccharides from LGTB- Neisseria meningitidis
US12/909,621 Expired - Fee Related US8221770B2 (en) 2002-08-02 2010-10-21 Vaccine compositions comprising L2 and/or L3 immunotype lipooligosaccharides from LGTB-neisseria meningitidis
US13/248,693 Abandoned US20120027800A1 (en) 2002-08-02 2011-09-29 Neisserial vaccine compositions comprising a combination of antigens
US13/299,007 Abandoned US20120064119A1 (en) 2002-08-02 2011-11-17 Neisserial vaccine compositions comprising a combination of antigens
US13/299,041 Abandoned US20120064120A1 (en) 2002-08-02 2011-11-17 Neisserial vaccine compositions comprising a combination of antigens
US14/844,989 Abandoned US20160082097A1 (en) 2002-08-02 2015-09-03 Neisserial vaccine compositions comprising a combination of antigens

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WO2004014418A2 (fr) 2004-02-19
ES2575014T3 (es) 2016-06-23
KR20050028051A (ko) 2005-03-21
KR20080078082A (ko) 2008-08-26
WO2004014417A3 (fr) 2004-07-22
IL166433A (en) 2011-08-31
PL375382A1 (en) 2005-11-28
NZ537181A (en) 2007-01-26
IS7658A (is) 2005-01-20
LU92262I2 (fr) 2013-09-30
US8221770B2 (en) 2012-07-17
JP5789203B2 (ja) 2015-10-07
EP1961427A3 (fr) 2009-11-04
US20060240045A1 (en) 2006-10-26
KR101237329B1 (ko) 2013-02-28
WO2004014417A2 (fr) 2004-02-19
KR20050042143A (ko) 2005-05-04
KR101139976B1 (ko) 2012-05-08
US20120027800A1 (en) 2012-02-02
TWI360424B (en) 2012-03-21
CA2489030A1 (fr) 2004-02-19
CN1671413A (zh) 2005-09-21
TW200408406A (en) 2004-06-01
EP2481419A2 (fr) 2012-08-01
PL220107B1 (pl) 2015-08-31
AU2003250204B8 (en) 2008-07-10
NO20050008L (no) 2005-04-28
IL165660A0 (en) 2006-01-15
US20120064120A1 (en) 2012-03-15
EP2255826A3 (fr) 2012-03-28
PL375408A1 (en) 2005-11-28
EP2258387A2 (fr) 2010-12-08
US20110033500A1 (en) 2011-02-10
JP4740738B2 (ja) 2011-08-03
EP2258384A3 (fr) 2011-12-28
AU2003250204B2 (en) 2008-06-19
PL375407A1 (en) 2005-11-28
IL166433A0 (en) 2006-01-15
WO2004015099A2 (fr) 2004-02-19
US20160082097A1 (en) 2016-03-24
PT2255826E (pt) 2016-06-08
EP2258385A3 (fr) 2012-01-18

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