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WO2011077143A1 - Transformation d'une neisseria commensale - Google Patents

Transformation d'une neisseria commensale Download PDF

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Publication number
WO2011077143A1
WO2011077143A1 PCT/GB2010/052179 GB2010052179W WO2011077143A1 WO 2011077143 A1 WO2011077143 A1 WO 2011077143A1 GB 2010052179 W GB2010052179 W GB 2010052179W WO 2011077143 A1 WO2011077143 A1 WO 2011077143A1
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sequence
nucleic acid
neisseria
seq
commensal neisseria
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Andrew Gorringe
Thomas Vaughan
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Public Health England
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Health Protection Agency
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • 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

Definitions

  • the present invention relates to methods and reagents for transforming commensal Neisseria such as Neisseria lactamica.
  • Infection by pathogenic organisms is one of the major causes of chronic and acute disease.
  • infection resulting from microbial sources such as bacteria, viruses and protozoa, continue to claim millions of lives worldwide.
  • microbial species increasingly becoming resistant to conventional antibiotics, it would be desirable to provide alternative and preferably prophylactic means of protecting against and fighting microbial infection.
  • Neisseria meningitidis is the causative agent of meningococcal meningitis and meningococcal septicaemia
  • Neisseria gonorrhoeae is the causative agent of gonorrhoea.
  • Neisseria lactamica (N. lactamica) closely resembles N. meningitidis, and both species are common inhabitants of the human nasopharynx. However, unlike N. meningitidis, N. lactamica lacks the key genes required for invasive disease and is limited to a commensal existence in the nasopharynx. Natural colonisation by N. lactamica is thought reduce the risk of invasive meningococcal disease, probably by inducing a cross-protective immune response.
  • N. lactamica is important as the basis for experimental live vaccines and outer- membrane vesicle (OMV) vaccines against meningococcal disease caused by N. meningitidis.
  • OMV outer- membrane vesicle
  • N. lactamica OMVs have been demonstrated to protect against lethal challenge in a mouse model of meningococcal disease (WO 00/50074).
  • OMVs from N. meningitidis have also been used in vaccines against meningococcal disease
  • OMVs from N. meningitidis and N. lactamica have been used in a vaccine blend (WO 03/051379).
  • N. lactamica it would be useful to be able to modify it genetically. Bacteria vary in their ability to take up and maintain foreign, heterologous DNA.
  • wild-type E. coli is resistant to transformation with DNA from non-E. coli sources, due to a restriction/ methylation system in which restriction endonucleases degrade foreign DNA while the cognate methylases protect "self DNA by methylating any recognition sites for the restriction endonucleases promptly after DNA synthesis.
  • Routine laboratory work therefore depends upon mutant strains of E. coli that are deficient in restriction endonuclease activity, for propagating and manipulating human or synthetic nucleic acids.
  • many natural isolates of N. meningitidis are amenable to genetic engineering using plasmids or linear DNA. Laboratory transformation of N. meningitidis may therefore be exploiting loopholes that have evolved in order to allow natural transfer of DNA between related bacteria.
  • natural isolates of N. lactamica resist stable uptake of foreign, heterologous DNA.
  • N. lactamica only two successful genetic modifications of N. lactamica are known: an NspA knockout and an NMB0033 (lytic murein transglycosylase A) knockout. These modified strains were obtained— at very low efficiencies— by disruptive insertion of antibiotic resistance genes. Such knockouts demonstrate that their targets are non-essential; however, attempts to insert larger DNA fragments into the N. lactamica chromosome have failed. Similarly, the rmpM gene of N. meningitidis can be knocked out and replaced with an extra copy of porA [Peeters et al. 1996, Vaccine (14); pages 1009-1015], but attempts to date to emulate this with N. lactamica have failed. Like wild-type E.
  • N. lactamica the transformation barrier in N. lactamica is believed to arise from a restriction/ methylation system in which restriction endonucleases degrade foreign, heterologous DNA while the cognate methylases protect "self DNA by methylating any recognition sites for the restriction endonucleases promptly after DNA synthesis.
  • N. lactamica restriction endonucleases At least five restriction endonucleases have been detected in N. lactamica, but only two of their cognate methylases are known, and no restriction-deficient mutants are available.
  • One of the N. lactamica restriction endonucleases, Nlal is an isoschizomer of the Haemophilus aegyptus enzyme Haelll. Nlal may share with Haelll an ability to cut single-stranded targets; if so, this may explain why conjugative transfer of single-stranded DNA has to date been unable to circumvent the N. lactamica transformation barrier. In this regard, conjugative transfer overcomes the transformation barrier in several organisms, but has failed with 53 isolates of N. lactamica [O'Dwyer CA et al. (2004), Infect. Immun., 72: pages 651 1 -6518].
  • N. lactamica eg. with a nucleotide sequence from a foreign, heterologous source.
  • an improved vaccine that provides protective immunity to infection by pathogenic organisms, such as pathogenic Neisseria (eg. N. meningitidis or N. gonorrhoea), or that is imnnunostinnulatory for the treatment of non-infectious disease, for example allergy and cancer.
  • the present invention provides a nucleic acid delivery vehicle, comprising an isolated polynucleotide sequence that is at least 300 nucleotides in length, wherein said polynucleotide sequence has at least 80% sequence identity to a nucleic acid sequence comprising at least 300 consecutive nucleotides of SEQ ID NO: 1 or 2; and wherein said polynucleotide sequence includes an insertion site into which a first nucleotide sequence may be inserted.
  • the present invention is based on homologous recombination of foreign DNA into the commensal Neisserial (eg. N. lactamica) chromosome.
  • the commensal Neisserial eg. N. lactamica
  • the foreign DNA sequence acquires the commensal host's methylation pattern; the foreign sequence is then protected from the host's restriction system, due to the requirement for the host to avoid self-restriction.
  • Nlad a prophage, termed 'Nlad ', residing in the genome of N. lactamica strain Y92-1009. Nlad is inserted into a region of the N. lactamica genome involved in respiration and the stress response. In N. lactamica strain Y92-1009, Nlad interrupts a serinyl-tRNA gene.
  • N. lactamica isolates contain the same (or closely related) prophage.
  • the prophage Nlad is a typical component of commensal Neisseria such as N. lactamica.
  • the presence of the prophage Nlad is of relevance not only to the commercially relevant N. lactamica strain Y92-1009, but also to many other (potentially all) N. lactamica strains and to other commensal Neisserial species such as Neisseria cinerea, Neisseria elongata, Neisseria flavescens, Neisseria polysaccharea, Neisseria sicca and Neisseria subflava.
  • the prophage Nlad has a 51562bp genome containing 65 genes. Sequence analysis by the inventors has identified that, in common with other bacteriophages, the Nlad sequence is divided into structural and regulatory regions, with genes for related functions clustered into contiguous blocks: the lysogeny block, the lytic growth block, the head-assembly block and the baseplate/ tail-assembly block (see Figure 1 ). The structural regions are passive during lysogeny.
  • Nlac1_024 and Nlac1_025 are two adjacent genes located within the tail-assembly block of the Nlad prophage.
  • the nucleic acid sequence of Nlac1_024 and Nlac1_025 is 100% identical, and is represented by SEQ ID NO: 3.
  • the Nlac1_024 and Nlac1_025 genes lie within a broader tandem-repeated region made up of two 1057 bp elements, named r601 and r602, which overlap each other by 39 base-pairs and are identical except for three nucleotide substitutions at the 3' end of r602.
  • r601 is found at nucleotide positions 18335-17279, and r602 is found at nucleotide positions 19353-18297 (numbering according to the coordinate system of Figure 1 , in which Nlac1_024 and Nlac1_025 are transcribed along the anticlockwise strand).
  • the nucleic acid sequence of r601 is represented by SEQ ID NO: 1 and the nucleic acid sequence of r602 represented by SEQ ID NO: 2.
  • the Nlac_024 coding sequence lies within r601 (see residues 359-1018 of SEQ ID NO: 1 ) and the Nlac_025 coding sequence lies within r602 (see residues 359-1018 of SEQ ID NO: 2).
  • Nlac1_024 and Nlac1_025 Due to the sequence identity of Nlac1_024 and Nlac1_025, these prophage genes are particularly attractive as sites for integration of foreign genetic material. In this regard, disruption of either one of the Nlac1_024 and Nlac1_025 genes is likely to leave the other gene intact to fulfil any unexpected functions that this pair of genes might have in maintaining lysogeny. Furthermore, due to the substantial identity of r601 and r602, a homologous recombination cassette constructed based on any of SEQ ID NOs: 1 , 2 or 3 (or a fragment thereof) effectively has a double target into which it can recombine.
  • a "commensal" organism coexists in an environment with another organism, such coexistence being beneficial to at least one of the organisms and generally not detrimental to either.
  • Neisseria lactamica Neisseria cinerea
  • Neisseria elongata Neisseria flavescens
  • Neisseria polysaccharea Neisseria sicca
  • Neisseria subflava Neisseria lactamica
  • Neisseria cinerea Neisseria cinerea
  • Neisseria elongata Neisseria flavescens
  • Neisseria polysaccharea Neisseria sicca
  • Neisseria subflava Neisseria subflava
  • Different commensal Neisseria species are known to colonise the buccal or nasal areas.
  • the commensal Neisseria is selected from Neisseria lactamica, Neisseria cinerea, Neisseria elongata, Neisseria flavescens, Neisseria polysaccharea, Neisseria sicca and Neisseria subflava.
  • the commensal Neisseria is Neisseria lactamica.
  • the commensal Neisseria is Neisseria lactamica strain Y92-1009.
  • a nucleic acid delivery vehicle is a nucleic acid construct comprising a "backbone” or “framework” polynucleotide sequence into which can be inserted a desired nucleotide sequence of interest.
  • the delivery vehicle of the invention comprises an isolated polynucleotide sequence that is at least 300 nucleotides in length, wherein said isolated polynucleotide sequence has at least 80% sequence identity to a nucleic acid sequence comprising at least 300 consecutive nucleotides of SEQ ID NO: 1 or 2; wherein said polynucleotide sequence comprises an insertion site into which may be inserted a first nucleotide sequence.
  • said isolated polynucleotide is at least 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025 or 1050 nucleotides in length.
  • said isolated polynucleotide is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 98% or 99% of the length of the nucleic acid sequence of SEQ ID NO: 1 or 2.
  • the polynucleotide sequence of SEQ ID NO: 1 or 2 may comprise one or more nucleic acid sequences that may function, under appropriate conditions, as an insertion site.
  • the isolated polynucleotide sequence comprises an insertion site that is naturally occurring in (ie. "endogenous" to) SEQ ID NO: 1 or 2.
  • the isolated polynucleotide sequence comprises an insertion site that does not occur naturally at that position in SEQ ID NO: 1 or 2.
  • the sequence of the isolated polynucleotide differs from the nucleic acid sequence of SEQ ID NO: 1 or 2 (or said at least 300 consecutive nucleotide region thereof).
  • the sequence of the polynucleotide is altered as compared with the nucleic acid sequence of SEQ ID NO: 1 or 2 (or said at least 300 consecutive nucleotide region thereof) so as to comprise said insertion site.
  • the isolated polynucleotide sequence in addition to any "endogenous" insertion site that occurs naturally at that position within SEQ ID NO: 1 or 2, also comprises an insertion site that does not occur naturally at that position in the nucleic acid sequence of SEQ ID NO: NO: 1 or 2.
  • the isolated polynucleotide sequence has less than 100% sequence identity to a nucleic acid sequence comprising at least 300 consecutive nucleotides of SEQ ID NO: 1 or 2.
  • the nucleic acid delivery vehicle comprises an isolated polynucleotide sequence that is at least 300 nucleotides in length, wherein said polynucleotide sequence has at least 80% sequence identity and less than 100% sequence identity to a nucleic acid sequence comprising at least 300 consecutive nucleotides of SEQ ID NO: 1 or 2; wherein said polynucleotide sequence comprises an insertion site into which may be inserted a first nucleotide sequence.
  • the delivery vehicle of the invention consists of an isolated polynucleotide sequence that is at least 300 nucleotides in length, wherein said polynucleotide sequence has at least 80% sequence identity to a nucleic acid sequence comprising (or consisting of) at least 300 consecutive nucleotides of SEQ ID NO: 1 or 2; and wherein said polynucleotide sequence includes an insertion site into which a first nucleotide sequence may be inserted.
  • the polynucleotide sequence has at least 85, 90, 92, 94, 95, 96, 97, 98 or 99% sequence identity to a nucleic acid sequence comprising (or consisting of) at least 300 consecutive nucleotides of SEQ ID NO: 1 or 2.
  • the polynucleotide may have at least about 99.5% or 9.8% sequence identity to a nucleic acid sequence comprising (or consisting of) at least 300 consecutive nucleotides of SEQ ID NO: 1 or 2.
  • the nucleic acid sequence of the isolated polynucleotide differs from the at least 300 consecutive nucleotides of SEQ ID NO: 1 or 2 at only about 100 nucleotide positions or fewer, such as at about 75, 50, 25, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotide positions. In one embodiment, the nucleic acid sequence of the isolated polynucleotide differs from the at least 300 consecutive nucleotides of SEQ ID NO: 1 or 2 at about 4, 3, 2 or 1 nucleotide positions.
  • any difference between the sequence of the isolated polynucleotide is selected from one or more nucleotide insertions, deletions and/ or substitutions.
  • the isolated polynucleotide sequence may have one or more nucleotide substitutions, as compared with the at least 300 consecutive nucleotides of SEQ ID NO: 1 or 2.
  • the nucleic acid sequence identity exists over the entire length of the nucleic acid sequence of SEQ ID NO: 1 or 2, or over the entire length of said at least 300 consecutive nucleotides thereof. Suitable conventional techniques for determining nucleic acid sequence identity are discussed below.
  • said isolated polynucleotide has at least 80% identity to a nucleic acid sequence comprising at least 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000 or 1025 consecutive nucleotides of SEQ ID NO: 1 or 2.
  • the at least 300 consecutive nucleotides of SEQ ID NO: 1 or 2 may be located anywhere within the sequence of SEQ ID NO: 1 or 2.
  • the consecutive stretch of at least 300 nucleotides may start from any nucleotide position from nucleotide 1 to nucleotide 757 of SEQ ID NO: 1 or 2.
  • the consecutive stretch of at least 300 nucleotides may end at any nucleotide position from nucleotide 300 to nucleotide 1057 of SEQ ID NO: 1 or 2.
  • said stretch of at least 300 consecutive nucleotides of SEQ ID NO: 1 or 2 starts at nucleotide residue 1 , 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725 or 750 of SEQ ID NO: 1 or 2.
  • said stretch of at least 300 consecutive nucleotides of SEQ ID NO: 1 or 2 ends at nucleotide residue 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025 or 1050 of SEQ ID NO: 1 or 2.
  • said isolated polynucleotide sequence has at least 80% sequence identity to a nucleic acid sequence comprising nucleotide residues 1 - 300, 50-350, 100-400, 150-450, 200-500, 250-550, 300-600, 350-650, 400-700, 450-800, 500-850, 600-900, 650-950, 700-1000 or 750-1050 of SEQ ID NO: 1 or 2.
  • said isolated polynucleotide sequence has at least 80% sequence identity to a nucleic acid sequence comprising (or consisting of) at least 300 consecutive nucleotides of SEQ ID NO: 1 or 2, wherein said at least 300 nucleotide nucleotides are from nucleotide residue 359 of SEQ ID NO: 1 or 2; or wherein said at least 300 nucleotide nucleotides end at nucleotide 1018 of SEQ ID NO: 1 or 2.
  • said isolated polynucleotide sequence has at least 80% sequence identity to a nucleic acid sequence comprising at least 300 consecutive nucleotides of SEQ ID NO: 3 (ie. residues 359-1018 of SEQ ID NOs: 1 and 2). In one embodiment, said isolated polynucleotide is at least 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 600, 625 or 650 nucleotides in length.
  • said isolated polynucleotide has a sequence length that is at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 98% or 99% of the length of the nucleic acid sequence of SEQ ID NO: 3.
  • the polynucleotide sequence of SEQ ID NO: 3 may comprise one or more nucleic acid sequences that may function, under appropriate conditions, as an insertion site.
  • the isolated polynucleotide sequence comprises an insertion site that is naturally occurring in (ie. "endogenous" to) SEQ ID NO: 3.
  • the isolated polynucleotide sequence comprises an insertion site that does not occur naturally at that position in SEQ ID NO: 3.
  • the sequence of the isolated polynucleotide differs from the nucleic acid sequence of SEQ ID NO: 3 (or said at least 300 consecutive nucleotide region thereof).
  • the sequence of the polynucleotide is altered as compared with the nucleic acid sequence of SEQ ID NO: 3 (or said at least 300 consecutive nucleotide region thereof) so as to comprise said insertion site.
  • the isolated polynucleotide sequence in addition to any "endogenous" insertion site that occurs naturally at that position within SEQ ID NO: 3, also comprises an insertion site that does not occur naturally at that position in the nucleic acid sequence of SEQ ID NO: NO: 3.
  • the isolated polynucleotide sequence has less than 100% sequence identity to a nucleic acid sequence comprising at least 300 consecutive nucleotides of SEQ ID NO: 3.
  • the nucleic acid delivery vehicle comprises an isolated polynucleotide sequence that is at least 300 nucleotides in length, wherein said polynucleotide sequence has at least 80% sequence identity and less than 100% sequence identity to a nucleic acid sequence comprising at least 300 consecutive nucleotides of SEQ ID NO: 3; wherein said polynucleotide sequence comprises an insertion site into which may be inserted a first nucleotide sequence.
  • the delivery vehicle of the invention consists of an isolated polynucleotide sequence that is at least 300 nucleotides in length, wherein said polynucleotide sequence has at least 80% sequence identity to a nucleic acid sequence comprising (or consisting of) at least 300 consecutive nucleotides of SEQ ID NO: 3; and wherein said polynucleotide sequence includes an insertion site into which a first nucleotide sequence may be inserted.
  • the polynucleotide sequence has at least 85, 90, 92, 94, 95, 96, 97, 98 or 99% sequence identity to a nucleic acid sequence comprising (or consisting of) at least 300 consecutive nucleotides of SEQ ID NO: 3.
  • the polynucleotide may have at least about 99.5% or 9.8% sequence identity to a nucleic acid sequence comprising (or consisting of) at least 300 consecutive nucleotides of SEQ ID NO: 3.
  • the nucleic acid sequence of the isolated polynucleotide differs from the at least 300 consecutive nucleotides of SEQ ID NO: 3 at only about 100 nucleotide positions or fewer, such as at about 75, 50, 25, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotide positions. In one embodiment, the nucleic acid sequence of the isolated polynucleotide differs from the at least 300 consecutive nucleotides of SEQ ID NO: 3 at about 4, 3, 2 or 1 nucleotide positions.
  • any difference between the sequence of the isolated polynucleotide is selected from one or more nucleotide insertions, deletions and/ or substitutions.
  • the isolated polynucleotide sequence may have one or more nucleotide substitutions, as compared with the at least 300 consecutive nucleotides of SEQ ID NO: 3.
  • the nucleic acid sequence identity exists over the entire length of the nucleic acid sequence of SEQ ID NO: 3, or over the entire length of said at least 300 consecutive nucleotides thereof. Suitable conventional techniques for determining nucleic acid sequence identity are discussed below.
  • said isolated polynucleotide has at least 80% identity to a nucleic acid sequence comprising at least 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640 or 650 consecutive nucleotides of SEQ ID NO: 3.
  • the isolated polynucleotide sequence of the invention comprises a site for insertion of a nucleotide sequence of interest.
  • the polynucleotide sequence comprises an insertion site that is naturally present in (or "endogenous to") the nucleic acid sequence of SEQ ID NO: 1 , 2 or 3.
  • nucleic acid sequences of SEQ ID NOs: 1 -2 and 3 comprise a number of naturally-occurring insertion sites, such as restriction sites.
  • the isolated polynucleotide sequence comprises residues 297-302 of SEQ ID NO: 1 or 2, residues 544-549 of SEQ ID NO: 1 or 2, residues 417-422 of SEQ ID NO: 1 or 2, and/ or residues 626-631 of SEQ ID NO: 1 or 2.
  • the isolated polynucleotide sequence comprises residues 59-64 of SEQ ID NO: 3, residues 186-191 of SEQ ID NO: 3, and/ or residues 268-273 of SEQ ID NO: 3.
  • an insertion site comprises a sequence of nucleic acid residues that is not naturally present at that location in the sequence of SEQ ID NO: 1 , 2 or 3.
  • Such an insertion site may conveniently be termed a “non-endogenous" insertion site, a “non-naturally occurring” insertion site, or an “artificial” insertion site.
  • the isolated polynucleotide sequence of the invention includes an insertion site that is not naturally present in (not “endogenous to”) the corresponding nucleic acid sequence of SEQ ID NO: 1 , 2 or 3.
  • the isolated polynucleotide sequence has less than 100% identity to a nucleic acid sequence comprising at least 300 consecutive nucleotides of SEQ ID NO: 1 , 2 or 3.
  • a non-naturally occurring insertion site may be introduced into a polynucleotide sequence by mutation or sequence modification (eg. genetic modification/ engineering), as compared with the nucleic acid sequence of SEQ ID NO: 1 , 2 or 3 (or said at least 300 consecutive nucleotides thereof).
  • An insertion site may be introduced by insertion of nucleotides into a polynucleotide sequence (as compared with the corresponding sequence of SEQ ID NO: 1 , 2 or 3).
  • an insertion site may be created in a polynucleotide sequence by deletion or substitution of one or more nucleotides of the corresponding sequence of SEQ ID NO: 1 , 2 or 3.
  • Conventional techniques are known in the art for genetically modifying a polynucleotide sequence by insertion, deletion and/ or substitution of one or more nucleotide residues.
  • the insertion site may be located anywhere in the polynucleotide sequence.
  • a skilled person is able to select a suitable location for the insertion site as a matter of routine. For example, selection of a suitable location to create an insertion site might be based on identifying a region of the polynucleotide sequence that requires little sequence modification (eg. only one nucleotide insertion, deletion or substitution) in order to create a nucleic acid sequence that comprises an insertion site.
  • insertion of the first nucleotide sequence into the delivery vehicle may depend on recognition of the nucleic acid sequence of the insertion site by an enzyme, such as a restriction enzyme or a recombinase enzyme.
  • the insertion site may comprise a nucleic acid sequence having single-stranded DNA ends
  • insertion of the nucleotide sequence of interest may comprise annealing between the single stranded ends of the insertion site and single-stranded ends of the nucleotide sequence of interest (eg. by ligation independent cloning, LIC).
  • the isolated polynucleotide sequence comprises an insertion site that comprises or consists of a nucleic acid sequence that is cleaved by a restriction enzyme.
  • Restriction enzymes are endonuclease enzymes that cleave the phosphodiester backbone of DNA. As such, restriction enzymes are also known as restriction endonucleases. A nucleic acid sequence that is cleaved by a restriction enzyme is known as a 'restriction site'.
  • Type II restriction enzymes By far the most common class of restriction enzymes used for cloning purposes are the Type II family. Most Type II restriction enzymes recognise and cleave a palindromic restriction site. However, Type ll(S) restriction enzymes recognise a specific non-palindromic asymmetric sequence (recognition sequence) and cleave a restriction site located at a specific distance from the recognition sequence.
  • the insertion site comprises or consists of a restriction site.
  • the restriction site is for a Type II restriction enzyme.
  • DNA restriction enzyme cleavage sequences have been well documented in the art, and corresponding DNA restriction enzymes that cleave said sequences are commercially available. It is therefore conventional for a skilled person to identify and select a suitable restriction site to ensure that a specific nucleotide sequence of interest may accurately be inserted into the delivery vehicle in the correct orientation.
  • a skilled person is also familiar with conventional techniques for engineering a restriction site into a polynucleotide sequence.
  • the insertion site may comprise or consist of a restriction site sequence that is cleaved by any of /Aval, AvrW, BamH ⁇ , Bell, BglW, BsePI, Bsgl, Btrl, EcoR ⁇ , Hind ⁇ , Nde ⁇ , Nhe ⁇ , Pst ⁇ , Sa/I, Spel, Xba ⁇ or mal.
  • the nucleic acid sequences that are recognised and cleaved by these restriction enzymes are provided below.
  • the insertion site comprises or consists of a BamHI restriction site.
  • a BamHI restriction site (GGATCC) can be generated in SEQ ID NO: 3 by a single base substitution from T to G at nucleotide position 197 of SEQ ID NO: 3 (corresponds to nucleotide position 555 of SEQ ID NOs: 1 and 2).
  • the insertion site comprises or consists of a Hindlll restriction site.
  • a Hindlll restriction site (AAGCTT) can be generated in SEQ ID NO: 3 by a single base substitution from G to C at nucleotide position 224 of SEQ ID NO: 3 (corresponds to nucleotide position 602 of SEQ ID NOs: 1 and 2).
  • the insertion site comprises or consists of a nucleic acid sequence that is recognised and cleaved by a recombinase enzyme.
  • the insertion site comprises or consists of single-stranded nucleic acid sequences.
  • the nucleotide sequence of interest is prepared with single-stranded nucleic acid overhangs (long 'sticky ends') that are complementary to the single-stranded nucleic acid sequences of the insertion site, and insertion of the nucleotide sequence of interest into the delivery vehicle is facilitated by complementary base pairing between the insertion site and the 'sticky ends' of the nucleotide sequence of interest.
  • the isolated polynucleotide sequence comprises more than one insertion site.
  • the polynucleotide sequence may comprise 2, 3, 4 or 5 insertion sites.
  • polynucleotide sequence permits accurate insertion of multiple (eg. 2, 3, 4 or 5) nucleotide sequences of interest into the delivery vehicle of the invention.
  • Each of the insertion sites may be selected from any of the types of insertion sites discussed above, and may be the same or may be different.
  • the at least two insertion sites in the isolated polynucleotide sequence are spaced apart by a stretch of at least 10, 20, 30 or 40 nucleotides, such as by about 45 nucleotides.
  • Said multiple insertion sites may be a mixture of endogenous insertion sites (ie. naturally occurring in the sequence of SEQ ID NOs: 1 , 2 or 3) and "non- endogenous"/ "engineered” insertion sites that do not occur naturally at that position in the sequence of SEQ ID NOs: 1 , 2 or 3.
  • multiple (eg. 2, 3, 4 or 5) insertion sites have been introduced (eg. engineered) into the sequence of said polynucleotide, as compared with the sequence of SEQ ID NO: 1 , 2 or 3, or said at least 300 consecutive nucleotides thereof.
  • said polynucleotide sequence comprises multiple non-naturally occurring/ artificial insertion sites, as compared with the corresponding nucleic acid sequence of SEQ ID NO: 1 , 2 or 3.
  • the multiple insertion sites comprise multiple restriction sites.
  • Each of the multiple restriction sites may comprise or consist of the same or different nucleic acid sequences, and may be cleaved by the same restriction enzyme or by different restriction enzymes.
  • the isolated polynucleotide sequence comprises more than one (eg. 2 or 3) engineered restriction sites that are cleaved by different restriction enzymes.
  • the polynucleotide sequence comprises a restriction site for a BamHI restriction endonuclease and a restriction site for another restriction endonuclease or recombinase.
  • the BamHI restriction site is not naturally occurring in the sequence of SEQ ID NOs: 1 , 2 or 3, and results from a single-nucleotide substitution (eg. as discussed above).
  • the other restriction site may be endogenous or non-endogenous to SEQ ID NOs: 1 , 2 or 3.
  • the polynucleotide sequence comprises a restriction site for a Hindlll restriction endonuclease and a restriction site for another restriction endonuclease or recombinase.
  • the Hindlll restriction site is not naturally occurring in the sequence of SEQ ID NOs: 1 , 2 or 3, and results from a single-nucleotide substitution (eg. as discussed above).
  • the other restriction site may be endogenous or non-endogenous to SEQ ID NOs: 1 , 2 or 3.
  • the polynucleotide comprises a BamHI restriction site and a Hindlll restriction site.
  • the BamHI and Hindlll restriction sites do not naturally occur at that position in the sequence of SEQ ID NOs: 1 , 2 or 3.
  • the isolated polynucleotide sequence has at least 80% sequence identity (eg. 85, 90, 92, 94, 95, 96, 97, 98, 99 or 100% sequence identity) to the nucleic acid sequence of SEQ ID NO: 1 or 2, and comprises a single base substitution from T to G at nucleotide position 555 as compared with the nucleic acid sequence of SEQ ID NO: 1 and 2, and a single base substitution from G to C at nucleotide position 602 as compared with the nucleic acid sequence of SEQ ID NO: 1 and 2.
  • sequence identity eg. 85, 90, 92, 94, 95, 96, 97, 98, 99 or 100% sequence identity
  • the isolated polynucleotide sequence has at least 80% sequence identity (eg. 85, 90, 92, 94, 95, 96, 97, 98, 99 or 100% sequence identity) to the nucleic acid sequence of SEQ ID NO: 3, and comprises a single base substitution from T to G at nucleotide position 197 as compared with the nucleic acid sequence of SEQ ID NO: 3 and a single base substitution from G to C at nucleotide position 224 as compared with the nucleic acid sequence of SEQ ID NO: 3.
  • sequence identity eg. 85, 90, 92, 94, 95, 96, 97, 98, 99 or 100% sequence identity
  • SEQ ID NO: 4 is an example of an isolated polynucleotide sequence of the invention.
  • SEQ ID NO: 4 represents the nucleic acid sequence of the 1057 bp element r601 (shown in SEQ ID NO: 1 ) but with a single-nucleotide substitution from T to G at nucleotide position 555 as compared with the nucleic acid sequence of SEQ ID NO: 1 , and a single-nucleotide substitution from G to C at nucleotide position 602 as compared with the nucleic acid sequence of SEQ ID NO: 1 .
  • the isolated polynucleotide sequence has at least 80% sequence identity (eg. 85, 90, 92, 94, 95, 96, 97, 98, 99 or 100% sequence identity) to a nucleic acid sequence comprising or consisting of at least 300 consecutive nucleotides of SEQ ID NO: 4 (eg.
  • polynucleotide sequence includes an insertion site into which may be inserted a first nucleotide sequence.
  • the delivery vehicle of the invention comprises or consists of a polynucleotide sequence having the nucleic acid sequence of SEQ ID NO: 4.
  • the invention also provides a plasmid comprising a nucleic acid delivery vehicle of the invention, as described above.
  • plasmids include vectors such as DNA vectors and RNA vectors.
  • the plasmid is a transcription vector for replication of the delivery vehicle in a host cell such as E. coli.
  • Plasmids typically contain control sequences such as an origin of replication and multi-cloning site, which are known to those skilled in the art and may be selected depending upon the host cells. Plasmids may also include a selectable marker.
  • the invention provides a host cell (such as E. coli) comprising a delivery vehicle or plasmid of the invention, as described above. The host cell replicates the delivery vehicle or plasmid.
  • the invention comprises propagating the delivery vehicle or plasmid of the invention in a host cell such as E. coli.
  • recombinant host cells refer to cells which can be, or have been, used as recipients for recombinant vector or other transfer DNA, and include the progeny of the original cell which has been transformed. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental or deliberate mutation.
  • the invention provides a method of producing a recombination cassette for integration of a first nucleotide sequence into the genome of a commensal Neisseria by homologous recombination, comprising inserting said nucleotide sequence of interest into a delivery vehicle of the invention as defined above.
  • a recombination cassette may be produced by inserting said first nucleotide sequence into a plasmid as defined above (which comprises said delivery vehicle of the invention).
  • a recombination cassette is prepared by inserting a first nucleotide sequence into the delivery vehicle or plasmid of the invention, via the insertion site.
  • a recombination cassette of the invention comprises a first nucleotide sequence, surrounded at both ends by flanking nucleic acid sequences from the delivery vehicle.
  • the polynucleotide sequence of the delivery vehicle is arranged as 2 flanking regions on either side of the inserted first nucleotide sequence.
  • the flanking region to the 5' of the first nucleotide sequence is known as the 5' flanking region
  • the flanking region to the 3' of the first nucleotide sequence is known as the 3' flanking region.
  • flanking nucleic acid sequences on either side of the first nucleotide sequence will vary, depending on the location of the insertion site within the polynucleotide sequence of the delivery vehicle.
  • the polynucleotide sequence may have at least 80% identity (but less than 100% identity) to SEQ ID NO: 1 or 2, with an insertion site located at position 555 as compared with the sequence of SEQ ID NOs: 1 or 2 into which the first nucleotide sequence is inserted.
  • the recombination cassette may comprise a 5' flanking region having about 555 nucleic acid residues of said polynucleotide sequence, and a 3' flanking region having about 502 nucleic acid residues of said polynucleotide sequence.
  • the polynucleotide sequence may have at least 80% identity (but less than 100% identity) to SEQ ID NO: 1 or 2, with an insertion site located at position 602 as compared with the sequence of SEQ ID NOs: 1 or 2 into which the first nucleotide sequence is inserted.
  • the recombination cassette may comprise a 5' flanking region having about 602 nucleic acid residues of said polynucleotide sequence, and a 3' flanking region having about 455 nucleic acid residues of said polynucleotide sequence.
  • nucleotides of the polynucleotide sequence of the delivery vehicle arranged on both sides of the insertion site (and hence on both sides of the inserted first nucleotide sequence in the recombination cassette).
  • the polynucleotide sequence may have at least 80% identity (but less than 100% identity) to SEQ ID NO: 3, with an insertion site located at position 224 as compared with the sequence of SEQ ID NOs: 3 into which the first nucleotide sequence is inserted.
  • the recombination cassette may comprise a 5' flanking region having about 224 nucleic acid residues of said polynucleotide sequence, and a 3' flanking region having about 76 nucleic acid residues of said polynucleotide sequence.
  • the polynucleotide sequence may have at least 80% identity (but less than 100% identity) to SEQ ID NO: 3, with an insertion site located at position 197 as compared with the sequence of SEQ ID NOs: 3 into which the first nucleotide sequence is inserted.
  • the recombination cassette may comprise a 5' flanking region having about 197 nucleic acid residues of said polynucleotide sequence, and a 3' flanking region having about 103 nucleic acid residues of said polynucleotide sequence.
  • the first nucleotide sequence may be cloned into the delivery vehicle or plasmid using a restriction enzyme that cleaves the restriction site.
  • a restriction enzyme that cleaves the restriction site.
  • the first nucleotide sequence may be cloned into the delivery vehicle or plasmid by site- specific recombination using a recombinase restriction enzyme that cleaves the recognition site.
  • a recombinase restriction enzyme that cleaves the recognition site.
  • the first nucleotide sequence is cloned into the insertion site of a plasmid-borne delivery vehicle, and the resulting recombination cassette is obtained from the plasmid.
  • the resulting recombination cassette is amplified from the plasmid-borne delivery vehicle (such as by PCR).
  • the method of producing a recombination cassette comprises inserting multiple (eg. 2 or more) nucleotide sequences of interest into said delivery vehicle or plasmid.
  • the resultant recombination cassette will therefore comprise multiple nucleotide sequences of interest (ie. "first", “second", “third” etc. nucleotide sequences).
  • the multiple nucleotide sequences may be inserted into the same insertion site, or may be inserted into different insertion sites.
  • the multiple nucleotide sequences of interest may be inserted into the delivery vehicle or plasmid substantially simultaneously, or they may be inserted sequentially (ie. one after the other).
  • the multiple nucleotide sequences inserted into the insertion site or sites may comprise the same nucleic acid sequence, or may comprise different nucleic acid sequences.
  • the multiple nucleic acid sequences may encode different polypeptides. Suitable nucleotide sequences of interest are discussed in more detail below.
  • the invention therefore provides a recombination cassette comprising a nucleic acid delivery vehicle of the invention, as described above, and further comprising a first nucleotide sequence inserted into said delivery vehicle via said insertion site.
  • said recombination cassette is obtained (or obtainable) by a method described above.
  • the recombination cassette may be circular, or may be non-circular.
  • the recombination cassette is a linear nucleic acid molecule (ie. having free 5' and 3' ends).
  • a linear nucleic acid sequence can be prepared from a closed circular molecule, such as a plasmid, by enzymatic digestion or physical disruption.
  • said recombination cassette comprises more than one nucleotide sequence of interest.
  • the recombination cassette may comprise 2 or more nucleotide sequences of interest, such as 3, 4 or 5 or more nucleotide sequences of interest.
  • the recombination cassette in addition to the first nucleotide sequence, may comprise a second nucleotide sequence and optionally a third, fourth or fifth (or further) nucleotide sequence inserted into one or more insertion sites.
  • the recombination cassette of the invention is capable of homologous recombination with genomic DNA of commensal Neisseria cells so as to introduce the nucleotide sequence(s) of interest into the commensal Neisseria cells' genomic DNA.
  • the recombination cassette of the invention is capable of "transforming" the commensal Neisseria cells.
  • a nucleotide sequence of interest sequence may comprise coding sequence (eg. one or more gene sequences), non-coding sequence (eg. regulatory sequences such as a promoter sequence or terminator sequence) or both coding and non- coding sequences.
  • coding sequence eg. one or more gene sequences
  • non-coding sequence eg. regulatory sequences such as a promoter sequence or terminator sequence
  • the nucleotide sequence of interest (eg. the first nucleotide sequence, as discussed above) comprises coding sequence.
  • the first nucleotide sequence may comprise one or more gene sequences, which encode one or more gene products.
  • the first nucleotide sequence comprises a gene sequence that encodes a peptide, polypeptide or protein, or a fragment thereof.
  • said first nucleotide sequence is a polycistronic nucleic acid sequence, comprising multiple (ie. at least two or more) polynucleotide sequences operably linked in the same reading frame.
  • the polycistronic nucleic acid sequence encodes a fusion protein.
  • the polycistronic nucleic acid sequence encodes multiple (ie. at least two or more) individual, separate polypeptide sequences.
  • the first nucleotide sequence comprises a nucleic acid sequence that is heterologous to said commensal Neisseria (ie. heterologous to the commensal Neisseria into which the nucleotide sequence of interest is to be integrated).
  • a polynucleotide sequence that is 'heterologous' to a commensal Neisseria is a polynucleotide sequence that is 'not native to' or 'not normally present in' or 'not naturally occurring in' the commensal Neisseria.
  • said heterologous polynucleotide sequence does not naturally occur in the genome of the commensal Neisseria.
  • the first nucleotide sequence comprises a coding sequence (eg. one or more gene sequences) that is heterologous to said commensal Neisseria.
  • the heterologous coding sequence may optionally be linked to non-coding sequence.
  • the non-coding sequence may be heterologous to said commensal Neisseria or may alternatively be native to (naturally occurring in) said commensal Neisseria.
  • Heterologous genes encode heterologous gene products.
  • the first nucleotide sequence comprises or consists of a gene that encodes a (at least one) gene product that is heterologous to said commensal Neisseria.
  • a gene product that is heterologous to a commensal Neisseria is a gene product that is 'not native to' the commensal Neisseria, or is 'not normally present in' or 'not naturally occurring in' the commensal Neisseria.
  • the gene product is not naturally encoded by the genome of the commensal Neisseria.
  • Examples of gene products that are heterologous to said commensal Neisseria include polypeptides, and fragments thereof, such as antigens or epitopes.
  • the first nucleotide sequence comprises a nucleic acid sequence that encodes a polypeptide, wherein said polypeptide is heterologous to said commensal Neisseria.
  • said polypeptide comprises an antigenic polypeptide, or an antigenic fragment thereof (eg. an epitope) that is heterologous to said commensal Neisseria.
  • the terms 'antigen' and 'antigenic polypeptide' are synonymous and mean any polypeptide that can be recognized by the immune system and/ or that induces an immune response in a host organism (eg. a mammal, such as a human) exposed to the antigenic polypeptide.
  • an antigenic polypeptide may stimulate a T-cell mediated immune response in the host organism and/ or may stimulate the generation of antibodies by the host organism.
  • an antigenic polypeptide is capable of specifically interacting with an antigen recognition molecule of the immune system, such as an immunoglobulin (antibody) or a T cell receptor.
  • an antigenic polypeptide may provide a cell-mediated response involving T cells (eg. CD4+ and/ or CD8+ T cells).
  • An antigenic polypeptide may have the ability to induce the secretion of Th1 -type cytokines such as IFN- ⁇ (eg. from predominantly CD4+ T cells).
  • An antigenic polypeptide comprises at least one antigenic determinant.
  • antigenic determinant and “epitope” are synonymous, and mean a part of an antigenic polypeptide that is recognised and bound by an antibody (or B cell or T cell) and elicits an immune response.
  • an antigenic polypeptide induces a neutralizing antibody response.
  • an antigenic polypeptide provides protection (such as long term protection) against subsequent challenge.
  • the first nucleotide sequence comprises a bacterial, viral, eukaryotic, fungal (eg. yeast) or protozoan nucleic acid sequence that is heterologous to said commensal Neisseria.
  • said heterologous bacterial, viral, eukaryotic, fungal or protozoan nucleic acid sequence encodes a bacterial, viral, eukaryotic, fungal (eg. yeast) or protozoan polypeptide, such as an antigenic polypeptide, that is heterologous to said commensal Neisseria.
  • said first nucleotide sequence comprises a nucleic acid sequence of a non-pathogenic (eg. commensal) organism, wherein said nucleic acid sequence is heterologous to said commensal Neisseria.
  • the first nucleotide sequence encodes a polypeptide of a non-pathogenic (eg. commensal) organism, wherein said polypeptide is heterologous to said commensal Neisseria.
  • said first polynucleotide sequence may comprise a nucleic acid sequence of a commensal Neisseria, wherein said nucleic acid sequence encodes a polypeptide that is heterologous to the commensal Neisseria into which the nucleotide sequence of interest is to be transformed.
  • the nucleic acid sequence may be from a different commensal Neisseria species or strain from the commensal Neisseria species or strain into which the nucleotide sequence of interest is to be transformed.
  • the heterologous gene may be a gene of Neisseria lactamica, Neisseria cinerea, Neisseria elongata, Neisseria flavescens, Neisseria polysaccharea, Neisseria sicca or Neisseria subflava.
  • said first nucleotide sequence comprises or consists of a nucleic acid sequence of a pathogenic (disease causing) organism. In one embodiment, said first nucleotide sequence encodes a polypeptide that comprises or consists of a polypeptide of a pathogenic (disease causing) organism.
  • pathogenic organisms examples include pathogenic bacteria, viruses, fungi (eg. yeast) and protozoa.
  • said first nucleotide sequence comprises or consists of a nucleic acid sequence encoding a polypeptide of a pathogenic bacterium (eg. of a gram positive bacterium or gram negative bacterium).
  • the first nucleotide sequence may encode a polypeptide comprising or consisting of a pathogenic bacterial polypeptide, such as a polypeptide of Acinetobacter, Actinobacillus, Actinomycetes, Aeromonas, Bacillus, Bordetella, Borrelia, Branhamella, Brucella, Calymmatobacterium, Campylobacter, Chlamydia, Clostridia, Corynebacterium, Coxiella, Enterobacter, Erwinia, Erysipelothrix, Escherichia, Francisella, Haemophilus, Klebsiella, Legionella, Leptospira, Listeria, Moraxella, Mycobacterium, Mycoplasma, Neisseri
  • said first nucleotide sequence comprises or consists of a nucleic acid sequence encoding a polypeptide of a pathogenic virus (eg. a viral surface peptide or glycoprotein).
  • a pathogenic viral polypeptide such as a polypeptide of polypeptide of rabies virus (eg. glycoprotein G), herpes simplex virus (eg. glycoprotein D), Epstein-Barr virus, vesicular stomatitis virus (eg. nudeoprotein), vaccinia virus, Human immunodeficiency virus (HIV), Hepatitis A virus (HAV), Hepatitis B (eg.
  • hepatitis B virus surface antigen Hepatitis C (HCV), human papillomavirus (HPV), Kaposi's Sarcoma-Associated Herpesvirus (KSHV), Respiratory Syncytial Virus, Ebola virus, Marburg virus, West Nile virus (WNV), St Louis Encephalitis virus (SLEV), Rift Valley Fever virus (RVFV), coronaviruses, rhinovirus, adenovirus, SIV, rotavirus, arbovirus, measles virus, polio virus, rubella virus, mumps virus, papova virus, varicella-zoster virus, varicella virus, hantavirus, arenavirus, bunyavirus, flavivirus, filovirus, cytomegalovirus, Tickborne hemorrhagic fever viruses, Tickborne encephalitis viruses and Influenza viruses.
  • said first nucleotide sequence comprises or consists of a nucleic acid sequence encoding a polypeptide of a pathogenic fungus (eg. a pathogenic yeast).
  • the first nucleotide sequence may encode a polypeptide comprising or consisting of a pathogenic fungal polypeptide, such as a polypeptide of Acremonium, Alternaria, Amylomyces, Arthoderma, Aspergillus, Aureobasidium, Blastochizomyces, Botrytis, Candida, Cladosporium, Crytococcus, Dictyostelium, Emmonsia, Fusarium, Geomyces, Geotrichum, Microsporum, Neurospora, Paecilomyces, Penicillium, Pilaira, Pityrosporum, Rhizopus, Rhodotorula, Saccharomyces, Stachybotrys, Trichophyton, Trichoporon or Yarrowia.
  • said first nucleotide sequence comprises or consists of a nucleic acid sequence encoding a polypeptide of a pathogenic protozoan.
  • the first nucleotide sequence may encode a polypeptide comprising or consisting of a pathogenic protozoan polypeptide, such as a polypeptide of Plasmodium falciparum; a trypanosome; or Cryptosporidium.
  • Said first nucleotide sequence may be up to about 10,000 nucleotides in length.
  • said first nucleotide sequence is up to about 7500, 5000, 4000, 3000, 2750, 2500, 2250, 2000, 1750, 1500, 1250 or 1000 nucleotides long.
  • said first nucleotide sequence may be in the region of about 100-3000 nucleotides long (eg. at least about 200, 300, 400, 500, 600, 700, 800, 900 or 1000 nucleotides in length).
  • said first nucleotide sequence comprises a nucleic acid sequence (eg. a gene) of a pathogenic Neisseria, such as a N. meningitidis or N. gonorrhoeae nucleic acid sequence.
  • the pathogenic Neisseria nucleic acid sequence encodes a pathogenic Neisseria polypeptide (eg. N. meningitidis or N. gonorrhoeae polypeptide). Examples of suitable pathogenic Neisseria polypeptides of interest (eg. N. meningitidis or N.
  • gonorrhoeae polypeptides include transferrin binding proteins, factor H binding proteins (fHbp), NadA, superoxide dismutase (such as Cu,Zn- SOD), Neisserial Surface Protein A (NspA), a Porin (eg. PorA or PorB), Opa, Opc, NhhA, or any other outer membrane protein of pathogenic Neisseria. Gene sequences for the majority of these antigens are known in the literature.
  • a nucleic acid sequence encoding a transferrin binding protein is provided by SEQ ID NO: 5; a nucleic acid sequence encoding a factor H binding proteins (fHbp) is provided by SEQ ID NO: 6; a nucleic acid sequence encoding a NadA is provided by SEQ ID NO: 7; a nucleic acid sequence encoding a Cu,Zn- SOD is provided by SEQ ID NO: 8; a nucleic acid sequence encoding a Neisserial Surface Protein A (NspA) is provided by SEQ ID NO: 9; a nucleic acid sequence encoding a PorA is provided by SEQ ID NO: 10; and a nucleic acid sequence encoding a PorB is provided by SEQ ID NO: 11.
  • NspA Neisserial Surface Protein A
  • the polynucleotide sequence of interest may comprise or consist of a nucleic acid sequence having at least 80% (eg. 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to any of SEQ ID NOs: 5-11 , or a fragment thereof comprising or consisting of at least about 21 consecutive nucleotides thereof (eg.
  • said first nucleotide sequence comprises a heterologous gene that encodes an immunostimulatory gene product, such as an immunostimulatory polypeptide.
  • An immunostimulatory gene product may be immunostimulatory for treatment of non-infectious disease, for example allergy or cancer.
  • an immunostimulatory polypeptide may comprise a nut antigen polypeptide (eg. peanut antigen), or may comprise a tumour-specific antigenic polypeptide (eg. melanoma-associated antigen "MAGE” or prostate specific antigen "PSA").
  • the embodiments of the invention as described above (and below) with respect to the first nucleotide sequence apply also to any other nucleotide sequence of interest that may be inserted into the delivery vehicle of the invention or that is inserted into the recombination cassette of the invention.
  • the recombination cassette of the invention comprises multiple inserted nucleotide sequences of interest (ie. "second”, “third”, “fourth” or “fifth” nucleotide sequences, etc.) the embodiments discussed above (and below) apply to these "second", “third”, “fourth” and "fifth” nucleotide sequences.
  • the recombination cassette of the invention further comprises one or more Neisserial Uptake Sequences.
  • a Neisserial Uptake Sequence is a short nucleic acid sequence (about 10 nucleotides in length) that promotes natural uptake of polynucleotide sequences by Neisseria sp.
  • An exemplary Neisserial Uptake Sequence comprises the nucleic acid sequence gccgtctgaa (shown in SEQ ID NO: 12), or the complement thereof.
  • the recombination cassette comprises a nucleic acid sequence having at least 80% sequence identity (eg. at least 85, 90, 95 or 100% sequence identity) to SEQ ID NO: 12, or a fragment thereof having at least 8 or 9 nucleotides.
  • the Neisserial Uptake Sequence may be naturally associated with the first nucleotide sequence.
  • the Neisserial Uptake Sequence is a part of the first nucleotide sequence.
  • the nucleic acid sequence encoding N. meningitidis Factor H binding protein (NMB1870) comprises a Neisserial Uptake Sequence at residues 3-12 of SEQ ID NO: 6.
  • the first nucleotide sequence does not inherently comprise a Neisserial Uptake Sequence (eg. if the first nucleotide sequence does not comprise a nucleic acid sequence of a Neisseria sp.), it is an option to modify the recombination cassette to include one or more Neisserial Uptake Sequences.
  • a Neisserial Uptake Sequence may be inserted into an insertion site of the recombination cassette.
  • the Neisserial Uptake Sequence may be inserted into the same insertion site as the first nucleotide sequence (eg. so it is located at the 5' or 3'end of the nucleotide sequence of interest).
  • the Neisserial Uptake Sequence may be inserted into a different insertion site from the site into which the first nucleotide sequence is inserted.
  • a Neisserial Uptake Sequence is inserted into the backbone of a plasmid vector that is used to propagate the recombination cassette prior to transformation into the intended recipient, or may be added to the ends of a linear recombination cassette by PCR amplification with a 10 bp tail on either one or both primers.
  • the first nucleotide sequence may be modified to include a Neisserial Uptake Sequence (eg. at the 5' or 3' end of the first nucleotide sequence) prior to inserting the first nucleotide sequence into the insertion site.
  • the recombination cassette of the invention may further comprise a nucleic acid 'signal sequence' encoding a 'signal peptide'.
  • a signal peptide is a short peptide that, as a component of a larger polypeptide (eg. a polypeptide encoded by the nucleotide sequence of interest), directs the polypeptide to a desired intracellular or extracellular location (such as the plasma membrane of a commensal Neisseria cell).
  • a desired intracellular or extracellular location such as the plasma membrane of a commensal Neisseria cell.
  • the first nucleotide sequence may comprise a gene sequence encoding a polypeptide operably linked to a nucleic acid 'signal sequence' encoding a signal peptide.
  • a signal sequence encoding a signal peptide is commonly positioned 5' to the first nucleotide sequence, although certain signal sequences may be positioned elsewhere in the first nucleotide sequence.
  • the signal peptide is naturally associated with the encoded polypeptide.
  • Neisseria PorA polypeptide is naturally produced with a signal peptide that translocates the nascent PorA polypeptide through the inner membrane.
  • the signal peptide may be native to the commensal Neisseria into which the recombination cassette is to be transformed (eg. an N. lactamica signal peptide).
  • the first nucleotide sequence does not encode a polypeptide comprising a signal peptide
  • a nucleic acid sequence encoding a signal peptide may be joined to the first nucleotide sequence in the correct reading frame.
  • the first nucleotide sequence is modified such that the encoded polypeptide is linked to a signal sequence from a different polypeptide, such as a commensal Neisseria polypeptide (eg. an N. lactamica polypeptide).
  • the recombination cassette comprises one or more non- coding sequences that drive or regulate the expression and processing of the first nucleotide sequence.
  • the non-coding sequence may be operably linked to the first nucleotide sequence (or may be comprised within the first nucleotide sequence, operably linked to a gene encoding a gene product of interest).
  • the first nucleotide sequence may comprise a nucleic acid sequence encoding a product of interest (eg. a polypeptide, as described above) operably linked (at the 5' or 3' end) to a leader sequence.
  • a leader sequence may affect processing of a primary DNA transcript to mRNA, and/ or may affect mRNA stability or translation efficiency.
  • the transcriptional and translational regulatory elements are functional in the commensal Neisseria into which the recombination cassette is transformed.
  • the transcriptional and translational regulatory elements are native to the commensal Neisseria.
  • the transcriptional and translational regulatory elements are naturally associated with the first nucleotide sequence.
  • operably linked means that the nucleic acid sequences being linked are contiguous and arranged so that they function in concert for their intended purposes - for example, transcription initiates in the promoter and proceeds through the coding polynucleotide segment to the terminator. Where necessary to join two protein coding regions, the polynucleotide coding sequences should be contiguous and in reading frame.
  • the recombination cassette comprises a selectable marker gene.
  • a selectable marker gene encodes an identifiable gene product, which enables identification of a cell that has been transformed with the recombination cassette.
  • the selectable marker gene encodes a protein necessary for the survival or growth of the transformed commensal Neisseria. This gene ensures the growth of only those commensal Neisseria that have been transformed.
  • Conventional selection genes encode proteins that (a) confer resistance to positive selection agents such as antibiotics or other toxic substances, eg. chloramphenicol, ampicillin, neomycin, methotrexate, etc.; (b) complement auxotrophic deficiencies; or (c) supply critical nutrients.
  • the first nucleotide sequence comprises or consists of a selectable marker gene.
  • recombination cassette comprises a first nucleotide sequence and at least one more nucleotide sequence, wherein one or more of said nucleotide sequences comprises or consists of a selectable marker gene.
  • the nucleotide sequence encoding the selectable marker gene may be inserted into the same insertion site as the first or other nucleotide sequence of interest.
  • the nucleotide sequence of interest encoding the selectable marker gene and the first (and/ or other) nucleotide sequence of interest may be inserted into the different insertion sites.
  • the invention provides a method of producing a recombination cassette for integration of multiple nucleotide sequences into the genome of a commensal Neisseria by homologous recombination, wherein at least one of said nucleotide sequences comprises or consists of a selectable marker gene, said method comprising inserting said nucleotide sequences of interest into a delivery vehicle of the invention or into a plasmid of the invention.
  • a selectable marker gene and a nucleotide sequence encoding an antigenic polypeptide are inserted into the delivery vehicle, to form a recombination cassette comprising both said selectable marker gene and said nucleotide sequence encoding an antigenic polypeptide.
  • the antigenic polypeptide may be, for example, a polypeptide of a pathogenic organism, such as a pathogenic Neisseria sp.
  • the first nucleotide sequence encodes N. meningitidis PorA polypeptide
  • the selectable marker gene is a chloramphenicol resistance gene (Cm R gene).
  • the invention provides a method of transforming commensal Neisseria with a first nucleotide sequence, comprising introducing a recombination cassette of the invention, as described above, into said commensal Neisseria.
  • Transformation' of commensal Neisseria refers to the heritable genetic alteration of commensal Neisseria cells resulting from the uptake, genomic incorporation, and expression of heterologous genetic material.
  • the commensal Neisseria is N. lactamica.
  • a skilled person is familiar with conventional techniques for introducing a recombination cassette into bacteria such as commensal Neisseria.
  • suitable techniques may be pilus-dependent (eg. natural uptake) or pilus- independent uptake (eg. via chemical transformation or electroporation).
  • the recombination cassette is capable of integration into the commensal Neisseria genome via homologous recombination.
  • the recombination cassette recombines with a region of the commensal Neisseria genome located within the Nlad prophage.
  • the recombination cassette targets the identical genes Nlac1_024 and Nlac1_025 (each represented by SEQ ID NO: 3), which are located within a tandem-repeated region of the prophage made up of two 1057 bp elements, named r601 (SEQ ID NO: 1 , comprising Nlac1_024) and r602 (SEQ ID NO: 2, comprising Nlac1_025).
  • the recombination cassette of the invention integrates into the genome of the commensal Neisseria (eg. N. lactamica) by homologous, site-specific recombination, resulting in a transformed, recombinant commensal Neisseria.
  • the commensal Neisseria eg. N. lactamica
  • the transformed, recombinant commensal Neisseria cells are known as "integrative transformants", because the heterologous nucleic acid sequence has become integrated into the genomic DNA of the commensal Neisseria cells.
  • the method further comprises selecting for transformed, recombinant commensal Neisseria that have stably integrated the first nucleotide sequence (and additional nucleotide sequence(s) of interest, if present in the recombination cassette) into their genome.
  • Neisseria colonies arising from the transformation may be screened directly for the presence of the first nucleotide sequence by PCR or by nucleic acid hybridisation. Such direct screening, though labour-intensive, has been found to be feasible in Neisseria spp. (Gunn JS & Stein DC 1996, Mol. Gen. Genet. 251 :509-17). Expression of a polypeptide encoded by the first nucleotide sequence may be verified using standard techniques such as Western blotting and flow-cytometry.
  • the first nucleotide sequence encodes a detectable polypeptide such as a reporter peptide or selectable marker (eg. as discussed above)
  • expression of the first nucleotide sequence may be verified by assaying for the reporter or selectable marker activity.
  • the selectable marker may protect the recombinant, transformed commensal Neisseria from the effects of a positive selection agent. Cells may be cultured in the presence of this positive selection agent. Transformed cells that have undergone homologous recombination will be protected against exposure to said positive selection agent via expression of said selectable marker.
  • the commensal Neisseria to be transformed is sensitive to an antibiotic or other toxic substance, and the selectable marker confers resistance to said antibiotic or toxic substance.
  • the commensal Neisseria is sensitive to chloramphenicol (ie. Cm s ) and the selectable marker gene provides resistance to chloramphenicol.
  • selecting for transformed, recombinant commensal Neisseria comprises selecting for commensal Neisseria that have developed resistance to said antibiotic or toxic substance - eg. commensal Neisseria that are Cm R .
  • Said selection method may comprise culturing the commensal Neisseria in the presence of said antibiotic or toxic substance (eg. on a medium comprising said antibiotic or toxic substance); growth of stably transformed, recombinant commensal Neisseria on this medium will be better than growth of commensal Neisseria that do not express the selectable marker.
  • the commensal Neisseria to be transformed has an auxotrophic deficiency, which is complemented by a product encoded by a nucleotide sequence of interest provided by the recombination cassette. Integrative transformation of the commensal Neisseria can be verified by detecting the substantial absence or reduction of the auxotrophic deficiency.
  • the invention provides a recombinant commensal Neisseria, such as Neisseria lactamica, that has a first nucleotide sequence of interest (eg. a heterologous gene sequence) stably integrated within its genome.
  • a first nucleotide sequence of interest eg. a heterologous gene sequence
  • the first nucleotide sequence is stably integrated into the genome of said recombinant commensal Neisseria within the commensal Neisseria Nlad prophage.
  • the first nucleotide sequence is stably integrated within the r601 or r602 tandem repeated region of the Nlad prophage, defined by SEQ ID NOs: 1 or 2. In one embodiment, the first nucleotide sequence is stably integrated within the Nlac_024 or Nlac_025 genes, defined by SEQ ID NO: 3 - ie. a ANIac1_024 foreign gene (or ANIac1_025::foreign gene) genotype.
  • the recombinant commensal Neisseria is obtainable (or has been obtained) by a transformation method as described above.
  • the integrated nucleotide sequence acquires the commensal Neisseria methylation pattern, and is therefore protected from the commensal Neisseria restriction system.
  • the recombinant commensal Neisseria expresses the first nucleotide sequence of interest.
  • said recombinant commensal Neisseria expresses a polypeptide (eg. as defined herein) encoded by the integrated first nucleotide sequence.
  • the polypeptide may comprise or consist of a heterologous polypeptide, such as an antigenic polypeptide, for example, a polypeptide of a pathogenic organism.
  • the invention provides a recombinant commensal Neisseria, such as N. lactamica, that expresses a polypeptide of a pathogenic Neisseria (eg. N. meningitidis or N. gonorroheae), wherein said polypeptide is encoded by a first nucleotide sequence that is stably integrated into the genome of said commensal Neisseria.
  • said recombinant commensal Neisseria such as N. lactamica, expresses an N.
  • said PorA coding sequence and said selectable marker gene are stably integrated within the Nlad prophage of said commensal Neisseria genome (eg. within the r601 or r602 sequence, such as within the Nlac_024 or Nlac_025 gene of said prophage - ie. a ANIac1_024 foreign gene genotype or ANIac1_025::foreign gene genotype).
  • the invention provides a method of transforming a commensal Neisseria, such as N. lactamica, with a first nucleotide sequence, comprising introducing into said commensal Neisseria chromosomal DNA obtained from a transformed, recombinant commensal Neisseria of the invention, as described above.
  • Chromosomal DNA from the genome of the transformed, recombinant commensal Neisseria has the correct commensal Neisseria methylation pattern and is therefore protected from the commensal Neisseria restriction enzymes. This enables high efficiency 'secondary transformation' of other, wild-type, commensal Neisseria.
  • Chromosomal DNA may be obtained from the transformed, recombinant commensal Neisseria using conventional techniques with which a skilled person is familiar. Likewise, suitable transformation techniques (pilus-dependent or pilus- independent) are also well known in the art - eg. as discussed above.
  • the invention also provides a 'secondarily' transformed, recombinant commensal Neisseria, obtainable by a method as described above.
  • Recombinant commensal Neisseria obtained by this secondary transformation method will not necessarily have ANIac1_024 foreign gene (or ANIac1_025::foreign gene) genotypes. Instead, the properly methylated DNA (including the nucleotide sequence of interest) may integrate into another locus in the commensal Neisseria genome.
  • the product of the first nucleotide sequence (eg. an antigenic polypeptide) is at least partially exposed at the surface of the commensal Neisseria cell.
  • the transformed, recombinant commensal Neisseria may be live, or may be killed.
  • commensal Neisseria may be killed using heat or by suspension in a mixture of bactericidal agents such as thiomersal and formaldehyde.
  • Live transformed commensal Neisseria may be attenuated. However, it is usually not required to attenuate commensal Neisseria because these organisms are avirulent.
  • An immunogenic component or extract may be obtained from the recombinant, commensal Neisseria of the invention, as described herein.
  • Said immunogenic component or extract may comprise a gene product (typically a polypeptide, such as an antigenic polypeptide) encoded by the first nucleotide sequence.
  • An immunogenic component or extract of the transformed, recombinant commensal Neisseria may comprise an outer membrane preparation, such as an outer membrane vesicle (OMV) preparation, or may comprise a protein fraction.
  • the outer membrane preparation (eg. OMVs) or protein fraction may comprise a gene product (typically a polypeptide, such as an antigenic polypeptide as described herein) encoded by the first nucleotide sequence.
  • Outer membrane vesicles are discrete vesicles formed or derived from fragments of the outer membrane of a Gram negative bacterium such as Neisseria (eg. commensal Neisseria such as N. lactamica).
  • OMVs typically comprise outer membrane proteins (OMPs), lipids, phospholipids, periplasmic material and lipopolysaccharide (LPS).
  • OMVs have a mean diameter of around 120nm and typically within the range of 80-200nm.
  • outer membrane components such as OMVs, protein fractions, lipooligosaccharides and lipopolysaccharides from cell preparations (see WO 00/50074), and are suitable to obtain the immunogenic components or extracts of the invention.
  • Outer membrane preparations and protein fractions can be obtained from commensal Neisseria cultured in the presence or absence of iron.
  • a protein fraction of commensal Neisseria is conveniently obtained by suspending commensal Neisseria cells or membranes in the presence of detergent and incubating the suspension so as to extract proteins therefrom.
  • OMVs can also be obtained from commensal Neisseria according to a number of methods known in the art, for example by deoxycholate extraction, Tris/HCI/EDTA extraction, and lithium acetate extraction. Protocols for performing such extractions are described in detail in the literature. However, it will be appreciated by the skilled person that virtually any chemical and/ or physical technique that enables disruption of the commensal Neisseria outer membrane in order to release sufficient OMVs for purification and isolation, is suitable for preparation of the compositions of the invention. Thus, in one aspect, the invention further provides a method of preparing an immunogenic component or extract of the recombinant, commensal Neisseria of the invention, as described herein.
  • the method comprises (i) suspending said recombinant commensal Neisseria in the presence of detergent; and (ii) incubating the suspension so as to extract an immunogenic component or extract from the recombinant commensal Neisseria.
  • Step (ii) of said method may further comprise the steps of (iii) centrifuging the suspension to separate the suspension into a supernatant and a pellet; and (iv) fractionating the an immunogenic component or extract from the supernatant.
  • This specific method can be modified according to the extraction protocol selected by the user.
  • alternative conventional techniques may be used, such as high salt concentration, chaotropic agents, high or low pH, enzymic digestion and/ or mechanical disruption.
  • the invention also provides an immunogenic composition, comprising a transformed, recombinant commensal Neisseria as described above, and optionally a pharmaceutically acceptable carrier.
  • the invention also provides an immunogenic composition
  • an immunogenic composition comprising an immunogenic component or extract of a transformed, recombinant commensal Neisseria of the invention, as described herein; and optionally a pharmaceutically acceptable carrier.
  • said immunogenic component or extract comprises a gene product (typically a polypeptide such as an antigenic polypeptide) encoded by said first nucleotide sequence.
  • said immunogenic component or extract comprises outer membrane vesicles.
  • the invention also provides a method of preparing an immunogenic composition, comprising combining a transformed, recombinant commensal Neisseria, as described above, with a pharmaceutically acceptable carrier.
  • the invention also provides a method of preparing an immunogenic composition, comprising: obtaining an immunogenic component or extract from a recombinant commensal Neisseria of the invention (eg. via a method as described above); and combining said immunogenic component or extract with a pharmaceutically acceptable carrier.
  • the immunogenic component or extract obtained from the recombinant commensal Neisseria comprises a polypeptide encoded by said first nucleotide sequence. In one embodiment, said immunogenic component or extract comprises outer membrane vesicles.
  • Non-limiting examples of pharmaceutically acceptable carriers include water, saline, and phosphate-buffered saline.
  • the composition is in lyophilized form, in which case it may include a stabilizer, such as BSA.
  • a preservative such as thiomersal or sodium azide, to facilitate long term storage.
  • immunogenic compositions of the invention comprise active immunogenic ingredients (ie. transformed, recombinant commensal Neisseria, or immunogenic component or extract thereof) and a pharmaceutically acceptable carrier, and may optionally comprise one or more of a excipient, diluent, adjuvant, buffering agent, immunoregulatory agent and/ or antimicrobial compound, as described below.
  • Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/ or adjuvants, which enhance the effectiveness of the vaccine.
  • adjuvants examples include but are not limited to: complete Freunds adjuvant (CFA), Incomplete Freunds adjuvant (IVA), Saponin, a purified extract fraction of Saporin such as Quil A, a derivative of Saporin such as QS-21 , lipid particles based on Saponin such as ISCOM/ISCOMATIX, E.
  • CFA complete Freunds adjuvant
  • IVA Incomplete Freunds adjuvant
  • Saponin a purified extract fraction of Saporin such as Quil A
  • QS-21 a derivative of Saporin
  • lipid particles based on Saponin such as ISCOM/ISCOMATIX
  • coli heat labile toxin (LT) mutants such as LTK63 and/ or LTK72, aluminium hydroxide, N- acetyl-muramyl-L-threonyl-D-isoglutamine ("thr-MDP"), N-acetyl-nor-muramyl-L- alanyl-D-isoglutamine (CGP 1 1637, "nor-MDP”), N-acetylmuramyl-L-alanyl-D- isoglutaminyl-L-alanine-2-(1 '-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryl oxy)- ethylamine (CGP 19835A, "MTP-PE”), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2 % squalene/ Tween
  • the vaccine of the invention may be "substantially free of adjuvant".
  • substantially free of adjuvant means that there is less than 0.05% adjuvant, such as less than 0.025% adjuvant, or less than 0.001 % adjuvant.
  • the vaccine may be completely free of adjuvant.
  • buffering agents include, but are not limited to, sodium succinate (pH 6.5), and phosphate buffered saline (PBS; pH 6.5 and 7.5).
  • the immunogenic composition (eg. vaccine) of the invention may further comprise one or more immunoregulatory agents selected from, for example, immunoglobulins, antibiotics, interleukins (eg. IL-2, IL-12), and/ or cytokines (eg. IFNy).
  • the immunogenic composition is a therapeutic or prophylactic formulation, or medicament, such as a vaccine.
  • a "vaccine” is a formulation that, when administered to a subject stimulates a protective immune response against infection or stimulates or desensitises the immune system in the treatment of a non-infectious medical condition such as an allergy or cancer.
  • the immune response may be a humoral and/ or cell-mediated immune response.
  • a vaccine of the invention can be used, for example, to protect a subject from the effects of infection by a pathogenic organism, such as Neisserial invention (eg. N. meningitidis or N. gonorrhoeae infection).
  • immunogenic compositions e.g. vaccines
  • administration of immunogenic compositions (eg. vaccines) of the invention to a subject is generally by conventional routes e.g. intravenous, subcutaneous, intraperitoneal, or mucosal routes.
  • the administration may be by parenteral injection, for example, a subcutaneous or intramuscular injection.
  • the therapeutic formulations, medicaments and prophylactic formulations (eg. vaccines) of the invention are typically prepared as injectables, either as liquid solutions or suspensions.
  • Solid forms suitable for solution in, or suspension in, liquid prior to injection may alternatively be prepared.
  • the preparation may also be emulsified, or the peptide encapsulated in liposomes or microcapsules.
  • An immunogenic composition comprising transformed commensal Neisseria of the invention that are buccal colonizers may be administered in a mouthwash.
  • Oral formulations may include conventional excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.
  • An immunogenic composition comprising transformed commensal Neisseria of the invention that are nasal colonizers may be administered in a nasal spray.
  • Formulations for intranasal administration may in the form of nasal droplets or a nasal spray.
  • An intranasal formulation may comprise droplets having approximate diameters in the range of 100-5000 ⁇ , such as 500-4000 ⁇ , 1000-3000 ⁇ or 100-1000 ⁇ .
  • the droplets may be in the range of about 0.001 -100 ⁇ , such as 0.1 -50 ⁇ or 1 .0-25 ⁇ , or such as 0.001 -1 ⁇ .
  • the immunogenic composition may be an aerosol formulation.
  • the aerosol formulation may take the form of a powder, suspension or solution. Aerosol particles may be delivered using a nebulizer (eg. via the mouth) or nasal spray. An aerosol formulation may optionally contain a propellant and/ or surfactant.
  • the size of aerosol particles is relevant to the delivery capability of an aerosol. Smaller particles may travel further down the respiratory airway towards the alveoli than would larger particles.
  • the aerosol particles have a diameter distribution to facilitate delivery along the entire length of the bronchi, bronchioles, and alveoli. Alternatively, the particle size distribution may be selected to target a particular section of the respiratory airway, for example the alveoli.
  • the particles may have diameters in the approximate range of 0.1 -50 ⁇ , such as 1 -25 ⁇ , or 1 -5 ⁇ .
  • Preferred dose ranges for administration of whole-cell transformed, recombinant commensal Neisseria are 0 ⁇ g to 100 ⁇ g.
  • Preferred dose ranges for administration of immunogenic components or extracts of said transformed, recombinant commensal Neisseria are from 0.2 g to 100 g.
  • the immunogenic composition (eg. vaccine) of the invention may contain 5% to 95% of active ingredient, such as at least 10% or 25% of active ingredient, or at least 40% of active ingredient or at least 50, 55, 60, 70 or 75% active ingredient.
  • the immunogenic composition may be administered in a single dose schedule, or in a multiple dose schedule.
  • a single dose schedule the full dose is given at substantially one time.
  • a multiple dose schedule is one in which a primary course of vaccination may be with 1 -10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or re-enforce the immune response, for example (for human subjects), at 1 -4 months for a second dose, and if needed, a subsequent dose(s) after a further 1 -4 months.
  • the dosage regimen will, at least in part, be determined by the need of the individual and be dependent upon the judgment of the practitioner.
  • the vaccine of the present invention may be administered as part of a 'prime-boost' vaccination regime.
  • Prime-boost vaccination regimes involve: Priming - ie. exposing a subject to one or more antigens or a vaccine; and subsequently: Boosting - ie. exposing the subject to one or more antigens or a vaccine.
  • the 'boost' antigen/ vaccine is typically different from the 'primer' antigen/ vaccine (known as "heterologous" prime-boost).
  • heterologous prime-boost immunization strategies have been shown to induce higher levels of effector T cell responses in subjects as compared with homologous boosting with the same vaccine.
  • the subject's immune system is 'primed' by administration of a conventional vaccine and then 'boosted' by administration of the vaccine of the present invention.
  • a subject's immune system may be 'primed' by administration of the vaccine of the present invention, and then 'boosted' by administration of a conventional vaccine.
  • the 'priming' step may be carried out on the subject at any age - in the case of mammalian subjects (eg. human subjects), priming is conventionally carried out neonatally, or during infancy, adolescence or adulthood.
  • the 'boosting' step may be carried out at any time after the 'priming' step.
  • a boosting step may be carried out at least about 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 weeks after the priming step, or at least about 3, 6, 8 or 12 months after the priming step, or at least about 2, 5, 10, 15, 20, 25, 30, 35, or 40 or more years after the boosting step.
  • the priming step is carried out during infancy and the boosting step is carried out during adolescence.
  • the immunogenic composition (eg. a vaccine) of the invention can be administered to a subject simultaneously or sequentially with one or more immunoregulatory agents selected from, for example, immunoglobulins, antibiotics, interleukins (eg. IL-2, IL-12), and/ or cytokines (eg. IFNy) and/ or one or more antimicrobial compounds, such as conventional anti-tuberculosis drugs (eg. rifampicin, isoniazid, ethambutol or pyrizinamide).
  • immunoregulatory agents selected from, for example, immunoglobulins, antibiotics, interleukins (eg. IL-2, IL-12), and/ or cytokines (eg. IFNy) and/ or one or more antimicrobial compounds, such as conventional anti-tuberculosis drugs (eg. rifampicin, isoniazid
  • the therapeutic formulation, medicament or prophylactic formulation (eg. a vaccine) is administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and/ or therapeutically effective.
  • an “effective amount” is a dosage or amount that is sufficient to achieve a desired biological outcome.
  • a “therapeutically effective amount” is an amount which is effective, upon single or multiple dose administration to a subject for treating, preventing, curing, delaying, reducing the severity of, ameliorating at least one symptom of a disorder or recurring disorder, or prolonging the survival of the subject beyond that expected in the absence of such treatment.
  • the quantity of active ingredient to be administered which is generally in the range of 5 micrograms to 250 micrograms of antigen per dose, depends on the subject to be treated, capacity of the subject's immune system to generate a protective immune response, and the degree of protection desired.
  • Precise amounts of active ingredient required to be administered may depend on the judgment of the practitioner and may be particular to each subject.
  • the transformed, recombinant commensal Neisseria, or immunogenic component or extract thereof, or immunogenic composition stimulates an immune response in a subject.
  • the invention provides a transformed, recombinant commensal Neisseria, or immunogenic component or extract thereof, or immunogenic composition, as described above, for use in stimulating an immune response in a subject.
  • the invention also provides the use of a transformed, recombinant commensal Neisseria, or immunogenic component or extract thereof, or immunogenic composition, as described above, for the manufacture of a medicament for stimulating an immune response in a subject.
  • the invention further provides a method for stimulating an immune response in a subject, comprising administering to the subject a transformed, recombinant commensal Neisseria, or immunogenic component or extract thereof, or immunogenic composition, as described above.
  • immune stimulation is measured by a protective effect in an in vivo survival assay.
  • immune stimulation is measured by an increased frequency in T lymphocytes specific for an antigen in the vaccine (ie. a T cell immune response).
  • the immune stimulation is a memory T cell immune response, such as a central memory T cell response (eg. a CCR7+ response).
  • immune stimulation is measured by an increase in antibody titer that is specific for an antigen in the vaccine.
  • the immune response is against a pathogenic organism, such as pathogenic Neisseria (eg. N. meningitidis or N. gonorrhoeae).
  • a pathogenic organism such as pathogenic Neisseria (eg. N. meningitidis or N. gonorrhoeae).
  • the invention provides a transformed, recombinant commensal Neisseria, or immunogenic component or extract thereof, or immunogenic composition, as described above, for use in treating or preventing an infection in a subject.
  • the invention also provides the use of a transformed, recombinant commensal Neisseria, or immunogenic component or extract thereof, or immunogenic composition, as described above, for the manufacture of a medicament for treating or preventing an infection in a subject.
  • the invention further provides a method for treating or preventing an infection in a subject, comprising administering to the subject a transformed, recombinant commensal Neisseria, or immunogenic component or extract thereof, or immunogenic composition, as described above.
  • infection includes the proliferation of a pathogenic organism within and/ or on the tissues of a host organism.
  • preventing an infection includes preventing the initiation of an infection and/ or reducing the severity or intensity of an infection.
  • treating an infection embraces therapeutic or preventative/ prophylactic measures, and includes post-infection therapy and amelioration of an infection.
  • the infection is a Neisse al infection - such as infection by N. meningitidis or N. gonorrhoeae.
  • administration of the recombinant commensal Neisseria, or immunogenic component or extract thereof, or immunogenic composition, as described above is useful for treating or preventing meningococcal disease. In one embodiment, administration of the recombinant commensal Neisseria, or immunogenic component or extract thereof, or immunogenic composition, as described above, is useful for treating or preventing meningitis or gonorrhoea.
  • the immune response is against an antigen, such as an allergen, or a disease (eg. tumour)-specific antigen.
  • an antigen such as an allergen, or a disease (eg. tumour)-specific antigen.
  • the invention provides a transformed, recombinant commensal Neisseria, or immunogenic component or extract thereof, or immunogenic composition, as described above, for use in stimulating or desensitizing the immune system in a subject.
  • the invention also provides the use of a transformed, recombinant commensal Neisseria, or immunogenic component or extract thereof, or immunogenic composition, as described above, for the manufacture of a medicament for stimulating or desensitizing the immune system in a subject.
  • the invention also provides a method for stimulating or desensitizing the immune system in a subject, comprising administering to the subject a transformed, recombinant commensal Neisseria, or immunogenic component or extract thereof, or immunogenic composition, as described above.
  • Methods and uses for stimulating or desensitizing the immune system in a subject may be useful for treating or preventing allergies (eg. nut allergies) or cancers.
  • the "efficacy" of a vaccine describes the ability of the vaccine to protect a subject (typically a mammalian subject eg. a human, bovine, porcine or equine subject) from challenge with a pathogen, or from a non-infectious medical condition.
  • vaccine efficacy may refer to the efficacy of a vaccine in preventing the initiation of an infection or non-infectious medical condition and/ or reducing the severity/ intensity of an infection or non-infectious medical condition.
  • An immunogenic composition of the invention may be administered to a subject already having an infection, or a condition or symptoms associated with an infection, to treat or prevent said infection.
  • the subject is suspected of having come in contact with an infectious organism, such as pathogenic Neisseria, or has had known contact with an infectious organism, but is not yet showing symptoms of exposure.
  • An immunogenic composition of the invention may be administered to a subject already having a non-infectious medical condition, or a condition or symptoms associated with a non-infectious medical condition, to treat or prevent said non-infectious medical condition.
  • the subject is suspected of having come in contact with an allergen, or has had known contact with an allergen, but is not yet showing symptoms of exposure.
  • the therapeutic composition/ medicament eg. vaccine
  • the therapeutic composition/ medicament may cure, delay, reduce the severity of, or ameliorate one or more symptoms, and/ or prolong the survival of a subject beyond that expected in the absence of such treatment.
  • a therapeutic/ prophylactic composition or medicament may be administered to a subject who ultimately may acquire an infection or a non-infectious medical condition, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of said infection or non-infectious medical condition, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • the subject has previously been exposed to an allergen or pathogen (eg. pathogenic Neisseria).
  • an allergen or pathogen eg. pathogenic Neisseria
  • the subject may have had an infection in the past (but is optionally not currently infected).
  • the subject may be latently infected with a pathogen.
  • the subject may have been vaccinated against the allergen or pathogen in the past.
  • the subject has been pre-exposed to a conventional vaccine against the allergen or pathogen in the past (eg. the subject's immune system has been 'primed').
  • a 'subject' is any animal subject that would benefit from stimulation of an immune response.
  • Typical animal subjects are mammals, for example, human, bovine, porcine, ovine, caprine, equine, corvine, canine or feline subjects.
  • the subject is human, bovine, porcine or equine.
  • Some pathogenic organisms such as pathogenic Neisseria are obligate human pathogens.
  • the 'subject' is a human.
  • the treatments and preventative therapies of the present invention are applicable to a variety of different subjects of different ages. In the context of humans, the therapies are applicable to children (eg.
  • the therapies are applicable to immature subjects and mature/ adult subjects.
  • the treatments and preventative therapies of the present invention are applicable to subjects who are immunocompromised or immunosuppressed (eg. human patients who have HIV or AIDS, or other animal patients with comparable immunodeficiency diseases), subjects who have undergone an organ transplant, bone marrow transplant, or who have genetic immunodeficiencies.
  • a neutralisation test examines the capability of antisera raised to a specific antigen to neutralise (ie. inhibit or prevent) a particular biological process associated with the functionality of the antigen.
  • antisera are raised to the antigen in an appropriate animal model (for example, mice, guinea pigs, rabbits, goats, sheep, horse) using the immunisation protocol that is appropriate to the host.
  • an appropriate animal model for example, mice, guinea pigs, rabbits, goats, sheep, horse
  • immunisation protocol that is appropriate to the host.
  • a standard immunisation protocol for guinea pigs might be immunisation with 0.5 nmoles antigen on day 1 , followed by two further 0.5 nmole inoculums over an eight-week period. Two weeks after the final dose, the sera would be obtained from the animals and used for neutralisation test.
  • a neutralisation test is typically performed by mixing the sera with the active antigen, or a protein containing the antigen, in vitro prior to assessment of the functionality of the antigen in an appropriate test.
  • a challenge test examines the capability of the antigen to raise a sufficient host response in order to neutralise (ie. inhibit or prevent) the pathogenesis of the agent from which the antigen is derived.
  • susceptible animals are inoculated with appropriate doses of the test antigen over an appropriate time period.
  • a standard immunisation protocol in mice might be initial immunisation with 5 g antigen on day 1 , followed by 5 g antigen on day 14 and 5 g antigen on day 28.
  • the animals are challenged with a test dose of the test agent and observed for susceptibility to the agent.
  • An antigen that demonstrated potential as a vaccine candidate would protect the animals from succumbing to the effects of the agent.
  • the challenge test therefore differs conceptually from the neutralisation test.
  • the neutralisation test assesses the ability of anti-antigen sera to inactivate an agent in vitro.
  • the challenge test assesses the ability of an antigen to raise a host response to the test agent - ie. the challenge test assesses in vivo efficacy.
  • nucleic acid sequence As used herein, the terms “nucleic acid sequence”, “nucleotide sequence”, “polynucleotide sequence” and “polynucleotide” are used interchangeably and do not imply any length restriction.
  • nucleic acid and “nucleotide” are also used interchangeably.
  • peptide and “protein” are used interchangeably and do not imply any length restriction.
  • Polynucleotide sequences include nucleic acid sequences that have been removed from their naturally occurring environment, recombinant or cloned DNA isolates, and chemically synthesized analogues or analogues biologically synthesized by heterologous systems.
  • isolated denotes that the polynucleotide sequence has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences (but may include naturally occurring 5' and 3' untranslated regions such as promoters and terminators), and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment. Methods for isolating nucleic acid sequences are known in the art.
  • polynucleotide means a polynucleotide of genomic, cDNA, semi-synthetic, or synthetic origin which, by virtue of its origin or manipulation: (1 ) is not associated with all or a portion of a polynucleotide with which it is associated in nature; or (2) is linked to a polynucleotide other than that to which it is linked in nature; and (3) does not occur in nature.
  • This artificial combination is often accomplished by via conventional chemical synthesis techniques, or by the artificial manipulation of isolated segments of nucleic acids - eg. by conventional genetic engineering techniques.
  • Polynucleotides may be prepared by any means known in the art. For example, large amounts of polynucleotides may be produced by replication in a suitable host cell such as E. coli. Natural or synthetic DNA fragments can be incorporated into recombinant nucleic acid constructs, typically DNA constructs, capable of introduction into and replication in a prokaryotic or eukaryotic cell.
  • Polynucleotides may also be produced by chemical synthesis, eg. by the phosphoramidite method or the triester method, and may be performed on commercial automated oligonucleotide synthesizers.
  • a double-stranded fragment may be obtained from the single stranded product of chemical synthesis either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
  • a nucleic acid sequence can be also obtained by conventional cloning procedures, such as PCR, or can be synthesized using nucleic acid synthesis machines.
  • An alternative way to prepare a full-length polynucleotide is to synthesize a specified set of overlapping oligonucleotides (eg. 40 to 100 nucleotides). Other sequences may be added that contain signals for proper initiation and termination of transcription and translation.
  • a "variant" nucleic acid sequence has substantial sequence homology or substantial sequence identity to a reference nucleic acid sequence (or a fragment thereof).
  • a nucleic acid sequence or fragment thereof is “substantially homologous" (or “substantially identical”) to a reference sequence if, when optimally aligned (with appropriate nucleotide insertions or deletions) with the other nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 80, 82, 84, 86, 88, 90, 92, 94, 96, 98 or 99% of the nucleotide bases.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences may be compared.
  • test and reference sequences are input into a computer, subsequent coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm then calculates the percentage sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • a nucleic acid sequence is substantially homologous (or substantially similar) to a corresponding naturally-occurring sequence when the two molecules are capable of hybridizing under selective hybridization conditions.
  • Selectivity of hybridization exists when hybridization occurs which is substantially more selective than total lack of specificity.
  • selective hybridization will occur when there is at least 80% homology over a stretch of at least about 300 nucleotides, such as at least about 80, 82, 84, 86, 88, 90, 92, 94, 96, 98 or 99% homology.
  • the length of homology comparison may be over longer stretches, and in certain embodiments will often be over a stretch of at least about 400, 500 or 600 nucleotides or more, for example over at least about 700, 800, 900 or 1000 or more nucleotides.
  • Nucleic acid hybridization will be affected by such conditions as salt concentration (eg. NaCI), temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art.
  • Stringent hybridisation conditions are preferably employed, and generally include temperatures in excess of 30°C, typically in excess of 37°C and preferably in excess of 45°C.
  • Stringent salt conditions will ordinarily be less than 1000 mM, typically less than 500 mM, and preferably less than 200 mM.
  • the pH is typically between 7.0 and 8.3. The combination of parameters is much more important than the measure of any single parameter.
  • a “fragment" of a polynucleotide comprises a series of consecutive amino acid residues from the sequence of said full-length polynucleotide.
  • a “fragment" of a polynucleotide may comprise (or consist of) at least 300 consecutive nucleic acid residues from the sequence of said polynucleotide (eg. at least 350, 400, 450, 500, 550, 600, 650, 700, 750, 800 850, 900, 950 or 1000 consecutive nucleic acid residues of said polynucleotide).
  • a fragment may include at least one antigenic determinant and/ or may encode at least one antigenic epitope of the corresponding polypeptide of interest.
  • Figure 1 shows a map of bacteriophage Nlad .
  • the two copies of the duplicated structural gene (Nlac1_024 and Nlac1_025) are shown as double-width arrowheads.
  • Nlad is represented in excised form for ease of illustration: the chromosomally integrated prophage is linearised by homologous recombination between attP and an identical 20 bp element in the N. lactamica chromosome.
  • Figure 2 shows a map of bacteriophage Nlad , with porA and Cm R integrated into Nlad 024.
  • Figure 3 shows a map of Nlac1_024, showing the positions of two engineered restriction sites, BamHI (G A GATC_C) and Hindlll (A A AGCT_T) - the altered nucleotides are shown in capitals. The nucleotide numbering is from 5' end of attP (the numbering in parentheses is for the identical gene Nlac1_025). Nlac1_024 and Nlac1_025 are on the negative strand. Note that Figure 3 is reverse- complemented relative to Figure 1 .
  • Figure 4 shows an alignment of the sequences of repeats r601 and r602 of the Nlad prophage.
  • the first and last codons of Nlac1_024 (within r601 ) and Nlac1_025 (within r602) are marked by their amino acid in bold.
  • the three nucleotide sequence differences between repeat r601 and repeat r602 are shown as white text in black boxes. Owing to the overlap between r601 and r602, the 39 nucleotides at the 5' end of r601 are also the 39 nucleotides at the 3' end of r602.
  • Figure 4 is reverse-complemented relative to Figure 1 .
  • Figure 5 shows flow-cytometric measurement of PorA surface-expression levels in four secondary recombinant clones of N. lactamica (each obtained by transformation with chromosomal DNA from a primary Nlac1_024-targeted recombinant clone). Data from N. meningitidis MC58 and from a AporA::KmR derivative of N. meningitidis H44/76 are included as positive and negative controls respectively.
  • Figure 6 shows SDS-PAGE (left) and western blot (right) of OMVs from N. meningitidis MC58, porA+ engineered N. lactamica Y92-1009 and wild-type N. lactamica.
  • the gel was stained with colloidal Coomassie Blue.
  • the blot was probed with monoclonal anti-PorA P1 .7, and visualised using an alkaline phosphatase-conjugated second antibody and AP New Magenta.
  • SEQ ID NO: 4 modified r601 with a BamHI and a Hind II I site arising from single-nucleotide substitutions at positions 555 and 602
  • SEQ ID NO: 5 coding sequence of transferrin binding protein A from
  • Neisseria meningitidis MC58 SEQ ID NO: 7 coding sequence of NadA (NMB1994) from Neisseria meningitidis MC58
  • NMB1398 from Neisseria meningitidis MC58 SEQ ID NO: 9 coding sequence of Neisserial surface protein A (NspA)
  • NMB0663 Neisseria meningitidis MC58
  • SEQ ID NO: 10 coding sequence of PorA (NMB1429) from Neisseria meningitidis MC58
  • SEQ ID NO: 11 coding sequence of PorB (NMB2039) from Neisseria meningitidis MC58
  • Example 1 Modification of the r601 element for use in recombination cassettes
  • the 1057 bp r601 element (SEQ ID NO: 1 ) was amplified by PCR and modified to contain two unique restriction sites, BamHI and Hindlll, 45 base pairs apart, by means of the single-nucleotide substitutions listed in Table 1 and illustrated in Figure 3.
  • the modified r601 element was propagated as an insert in an E. coli plasmid.
  • Cm R chloramphenicol resistance
  • porA 42 kDa porin
  • the resulting recombination cassette was amplified from the plasmid using the same primer-pair that was previously used to amplify r601 from N. lactamica.
  • the linear cassette was transformed into N. lactamica (Cm s porA " ) and recombinant colonies were obtained by selection on TSA-yeast agar containing 0-5 g ml -1 chloramphenicol.
  • PorA by the recombinant N. lactamica was verified by Western blotting and flow-cytometry.
  • Example 3 secondary transfer of porA and CmR genes from the primary recombinant N. lactamica into wild-type N. lactamica
  • N. lactamica strains obtained this way do not necessarily have NIac1_024 ⁇ foreign gene (or NIac1_025 foreign gene) genotypes.
  • Properly methylated DNA may integrate into other loci.
  • Transformed N. lactamica strain Y92-1009 (prepared according to Example 2 or 3 above) is grown in Mueller Hinton broth (MHB) containing 5 gml "1 ethylenediamine- N, N'bis(2-hydroxyphenylacetic acid) (EDDHA), and incubated at 37°C with shaking (140rpm) for approximately 6h.
  • MHB Mueller Hinton broth
  • EDDHA ethylenediamine- N, N'bis(2-hydroxyphenylacetic acid)
  • PBS phosphate buffered saline
  • % (v/v) formaldehyde and 0.1 % (w/v) thiomersal Bacteria are then harvested by centrifugation and resuspended in phosphate buffered saline (PBS) containing 1 % (v/v) formaldehyde and 0.1 % (w/v) thiomersal, and left to stand overnight at 2-8°C.
  • Killed cells are then resuspended in PBS to an OD 6 5o of 1 .0 (equivalent to 2 x 10 9 CFUml "1 ) and alhydrogel is added to 25% (V/V), yielding a product suitable for subcutaneous administration.
  • This method is suitable also for N. cinerea, N. elongata, N. flavescens, N. polysaccharea, N. sicca and N. subflava.
  • Transformed N. lactamica strain Y92-1009 (prepared as per Example 2 or 3) is grown in MHB with and without the addition of 5 gmr 1 EDDHA overnight at 37°C with shaking. Iron limited (with EDDHA) and iron replete cells were then treated separately. Bacteria from 1 .5 litres are harvested by centrifugation and resuspended in 20ml 200mM Lithium acetate, 5mM EDTA, pH 6.0 and incubated for 3h at 37°C with shaking. Bacteria are then passed 7 times through a 21 gauge needle and pelleted at 8000g for 10m in. The supernatant is recovered and membranes pelleted by centrifugation at 100,000g for 1 h at 4°C.
  • OM-containing vaccinating preparations The membranes are then resuspended in 10mM HEPES, pH 7.4, containing 0.1 % (v/v) 10mM PMSF, yielding OM-containing vaccinating preparations.
  • the protein content of the OM vaccine preparations is determined using the bicinchoninic acid assay (Sigma, UK).
  • OMs are diluted in sterile deionized water to give a protein concentration of l OO gml "1 . This is then mixed with an equal volume of Alhydrogel, to give a final protein concentration of 50 gml "1 , and emulsified thoroughly.
  • Alhydrogel Superfoss, Denmark) is used for the primary dose, and for subsequent boosts.

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Abstract

La présente invention concerne des procédés et des réactifs, spécialement des véhicules de distribution d'acide nucléique, pour la transformation d'une Neisseria commensale telle que la Neisseria lactamica. Ceci est fondé sur les régions de répétition en tandem r601 (SEQ ID NO: 1) et r602 (SEQ ID NO: 2) du prophage Nlacl intégré de façon chromosomique de Neisseria et sur deux gènes identiques Nlacl_024 et Nlacl_025 (SEQ ID NO: 3) présents dans lesdites régions r601 et r602. L'invention concerne un véhicule de distribution d'acide nucléique, comportant une séquence polynucléotidique isolée qui est d'au moins 300 nucléotides de longueur, ladite séquence polynucléotidique ayant au moins 80 % d'identité de séquence par rapport à une séquence d'acide nucléique comportant au moins 300 nucléotides consécutifs de SEQ ID NO: 1 ou 2 ; ladite séquence polynucléotidique comprenant un site d'insertion dans lequel une première séquence nucléotidique peut être insérée.
PCT/GB2010/052179 2009-12-21 2010-12-21 Transformation d'une neisseria commensale Ceased WO2011077143A1 (fr)

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WO2016036839A1 (fr) * 2014-09-02 2016-03-10 The Arizona Board Of Regents On Behalf Of The University Of Arizona Compositions et méthodes de traitement de la gonorrhée
WO2017103593A1 (fr) * 2015-12-15 2017-06-22 University Of Southampton Infection à méningocoque et bactérie du genre neisseria lactamica modifiée
US10900043B2 (en) 2014-09-02 2021-01-26 Arizona Board Of Regents On Behalf Of The University Of Arizona Compositions and methods for treating bacterial disease

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Publication number Priority date Publication date Assignee Title
WO2016036839A1 (fr) * 2014-09-02 2016-03-10 The Arizona Board Of Regents On Behalf Of The University Of Arizona Compositions et méthodes de traitement de la gonorrhée
US10286016B2 (en) 2014-09-02 2019-05-14 Arizona Board Of Regents On Behalf Of The University Of Arizona Compositions and methods for treating gonorrhea
US10900043B2 (en) 2014-09-02 2021-01-26 Arizona Board Of Regents On Behalf Of The University Of Arizona Compositions and methods for treating bacterial disease
WO2017103593A1 (fr) * 2015-12-15 2017-06-22 University Of Southampton Infection à méningocoque et bactérie du genre neisseria lactamica modifiée
US20190307874A1 (en) * 2015-12-15 2019-10-10 University Of Southampton Meningococcal infection and modified neisseria lactamica
US10960067B2 (en) 2015-12-15 2021-03-30 University Of Southampton Meningococcal infection and modified Neisseria lactamica

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