EP1848457A2 - Vaccines against neisseria meningitidis - Google Patents

Vaccines against neisseria meningitidis

Info

Publication number
EP1848457A2
EP1848457A2 EP05823115A EP05823115A EP1848457A2 EP 1848457 A2 EP1848457 A2 EP 1848457A2 EP 05823115 A EP05823115 A EP 05823115A EP 05823115 A EP05823115 A EP 05823115A EP 1848457 A2 EP1848457 A2 EP 1848457A2
Authority
EP
European Patent Office
Prior art keywords
protein
dna
polypeptide
sequence
nmb
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP05823115A
Other languages
German (de)
French (fr)
Inventor
Christoph Marcel Tang
Yanwen Li
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ip2ipo Innovations Ltd
Original Assignee
Imperial Innovations Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/GB2004/005441 external-priority patent/WO2005060995A2/en
Application filed by Imperial Innovations Ltd filed Critical Imperial Innovations Ltd
Publication of EP1848457A2 publication Critical patent/EP1848457A2/en
Withdrawn legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/22—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Neisseriaceae (F)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention relates to vaccines and their use, and in particular to vaccines for meningococcal disease.
  • Microbial infections remain a serious risk to human and animal health, particularly in light of the fact that many pathogenic microorganisms, particularly bacteria, are or may become resistant to anti-microbial agents such as antibiotics.
  • Vaccination provides an alternative approach to combating microbial infections, but it is often difficult to identify suitable imrnunogens for use in vaccines which are safe and which are effective against a range of different isolates of a pathogenic microorganism, particular a genetically diverse microorganism.
  • vaccines which use as the immunogen substantially intact microorganisms, such as live attenuated bacteria which typically contain one or mutations in a virulence-deterrnining gene, not all microorganisms are amenable to this approach, and it is not always desirable to adopt this approach for a particular microorganism where safety cannot always be guaranteed.
  • some microorganisms express molecules which mimic host proteins, and these are undesirable in a vaccine.
  • Neisseria meningitidis which causes meningococcal disease, a life threatening infection which in the Europe, North America, developing countries and elsewhere remains an important cause of childhood mortality despite the introduction of the conjugate serogroup C polysaccharide vaccine.
  • infections caused by serogroup B strains (NmB) which express an cc2-8 linked polysialic acid capsule, are still prevalent.
  • serogroup in relation to N. meningitidis refers to the polysaccharide capsule expressed on the bacterium.
  • the common serogroup in the UK causing disease is B, while in Africa it is A.
  • Meningococcal septicaemia continues to carry a high case fatality rate; and survivors are often left with major psychological and/or physical disability. After a non-specific prodromal illness, meningococcal septicaemia can present as a fulminant disease that is refractory to appropriate anti-microbial therapy and full supportive measures. Therefore, the best approach to combating the public health menace of meningococcal disease is through prophylactic vaccination.
  • meningitidis infections are based on the polysaccharide capsule located on the surface of bacterium (Anderson et al (1994) Safety and immunogenicity of meningococcal A and C polysaccharide conjugate vaccine in adults. Infect Immun.
  • the most validated immunologic correlate of protection against meningococcal disease is the serum bactericidal assay (SBA).
  • SBA serum bactericidal assay
  • the SBA evaluates the ability of antibodies (usually IgG2a subclass) in serum to mediate complement deposition on the bacterial cell surface, assembly of the membrane attack complex, and bacterial lysis.
  • a known number of bacteria are exposed serial dilutions of the sera with a defined complement source. The number of surviving bacteria is determined, and the SBA is defined as the reciprocal of the highest dilution of serum that mediates 50% killing.
  • the SBA is predictive of protection against serogroup C infections, and has been widely used as a surrogate for immunity against NmB infections.
  • the SBA is a ready marker of immunity for the pre-clinical assessment of vaccines, and provides a suitable endpoint in clinical trials.
  • the key to a successful vaccine is to define antigen(s) that elicit protection against a broad range of disease isolates irrespective of serogroup or clonal group.
  • a genetic screening method (which we have termed Genetic Screening for Immunogens or GSI) was used to isolate antigens that are conserved across the genetic diversity of microbial strains and this is exemplified in relation to meningococcal strains.
  • the GSI method relates to a method for identifying a polypeptide of a microorganism which polypeptide is associated with an immune response in an animal which has been subjected to the microorganism, the method comprising the steps of (1) providing a plurality of different mutants of the microorganism; (2) contacting the plurality of mutant microorganisms with antibodies from an animal which has raised an immune response to the microorganism or a part thereof, under conditions whereby if the antibodies bind to the mutant microorganism the mutant microorganism is killed; (3) selecting surviving mutant microorganisms from step (2); (4) identifying the gene containing the mutation in any surviving mutant microorganism; and (5) identifying the polypeptide encoded by the gene. It will be appreciated that by the way in which the polypeptides have been identified, they are highly relevant as antigenic polypeptides.
  • genes identified by the GSI method are the NBM0341 (TspA), NMB0338, NMB1345, NMB0738, NBM0792 (NadC family), NMB0279, NMB2050, NMB1335 (CreA), NMB2035, NMB1351 (Fmu and Fmv), NMB1574 (HvC), NMB1298 (rsuA), NMB1856 (LysR family), NMBOl 19, NMB1705 (rfak), NMB2065 (HemK), NMB0339, NMB0401 (putA), NMB 1467 (PPX), NMB2056, NMB0808, NMB0774 (upp), NMA0078, NMB0337 (branched-chain amino acid transferase), NMB0191 (ParA family), NMB1710 (glutamate dehydrogenase (gdhA), NMB0062 (rfbA-1), NMB1583 (MsB), NBM0341 (TspA),
  • these genes form part of the genome that has been sequenced, as far as the inventors are aware, they have not been isolated, the polypeptides they encode have not been produced (and have not been isolated), and there is no indication that the polypeptides they encode may be useful as a component of a vaccine.
  • the invention includes the isolated genes as above and in the Examples and variants and fragments and fusions of such variants and fragments, and includes the polypeptides that the genes encode as described above, along with variants and fragment thereof, and fusions of such fragments and variants. Variants, fragments and fusions are described hi more detail below.
  • the variants, fragments and fusions of the given genes above are ones which encode a polypeptide which gives rise to neutralizing antibodies against N. meningitidis.
  • the variants, fragments and fusions of the polypeptide whose sequence is given above are ones which gives rise to neutralizing antibodies against N. meningitidis.
  • the neutralising antibodies may be produced in any animal with an immune system, for example a rat, mouse or rabbit.
  • the invention also includes isolated polynucleotides encoding the polypeptides whose sequences are given in the Example (preferably the isolated coding region) or encoding the variants, fragments or fusions.
  • the invention also includes expression vectors comprising such polynucleotides and host cells comprising such polynucleotides and vectors (as is described in more detail below).
  • the polypeptides described in the Examples are antigens identified by the method of the invention.
  • Variants of the gene may be made, for example by identifying related genes in other microorganisms or in other strains of the microorganism, and cloning, isolating or synthesizing the gene.
  • variants of the gene are ones which have at least 70% sequence identity, more preferably at least 85% sequence identity, most preferably at least 95% sequence identity with the genes as given above.
  • replacements, deletions and insertions may be tolerated.
  • the degree of similarity between one nucleic acid sequence and another can be determined using the GAP program of the University of Wisconsin Computer Group.
  • Variants of the gene are also ones which hybridise under stringent conditions to the gene.
  • stringent we mean that the gene hybridises to the probe when the gene is immobilised on a membrane and the probe (which, in this case is >200 nucleotides in length) is in solution and the immobilised gene/hybridised probe is washed in 0.1 x SSC at 65°C for 10 min. SSC is 0.15 M NaCl/0.015 M Na citrate.
  • Fragments of the gene may be made which are, for example, 20% or 30% or 40% or 50% or 60% or 70% or 80% or 90% of the total of the gene.
  • Preferred fragments include all or part of the coding sequence.
  • the variant and fragments may be fused to other, unrelated, polynucleotides.
  • the polynucleotide encodes a polypeptide which is immunogenic and is reactive with the antibodies from an animal which has been subjected to the microorganism from which the gene was identified.
  • the antigen may be the polypeptide as encoded by the gene identified above, and the sequence of the polypeptide may readily be deduced from the gene sequence.
  • the antigen may be a fragment of the identified polypeptide or may be a variant of the identified polypeptide or may be a fusion of the polypeptide or fragment or variant.
  • a particular aspect of the invention provides a polypeptide comprising the amino acid sequence selected from any one of SEQ ID Nos 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68; or a fragment or variant thereof or a fusion of such a fragment or variant.
  • the invention provides the following isolated proteins, or fragments or variants thereof, or fusion of these: NMB0341, NMB 1583,
  • NMB1345 NMB0738, NMB0792, NMB0279, NMB2050, NMB1335,
  • Fragments of the identified polypeptide may be made which are, for example, 20% or 30% or 40 % or 50% or 60% or 70% or 80% or 90% of the total of the polypeptide. Typically, fragments are at least 10, 15, 20, 30, 40 , 50, 100 or more amino acids, but less than 500, 400, 300 or 200 amino acids.
  • Variants of the polypeptide may be made. By “variants” we include insertions, deletions and substitutions, either conservative or non-conservative, where such changes do not substantially alter the normal function of the protein. By “conservative substitutions” is intended combinations such as GIy, Ala; VaI, He, Leu; Asp, GIu; Asn, GIn; Ser, Thr; Lys, Arg; and Phe, Tyr. Such variants may be made using the well known methods of protein engineering and site-directed mutagenesis.
  • variants are those encoded by variant genes as discussed above, for example from related microorganisms or other strains of the microorganism.
  • variant polypeptides typically have at least 70% sequence identity, more preferably at least 85% sequence identity, most preferably at least 95% sequence identity with the polypeptide identified using the method of the invention.
  • the percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequence has been aligned optimally.
  • the alignment may alternatively be carried out using the Clustal W program (Thompson et al, (1994) Nucleic Acids Res 22, 4673-80). The parameters used may be as follows:
  • Fast pairwise alignment parameters K-tuple(word) size; 1, window size; 5, gap penalty; 3, number of top diagonals; 5. Scoring method: x percent.
  • the fusions may be fusions with any suitable polypeptide.
  • the polypeptide is one which is able to enhance the immune response to the polypeptide it is fused to.
  • the fusion partner may be a polypeptide that facilitates purification, for example by constituting a binding site for a moiety that can be immobilised in, for example, an affinity chromatography column.
  • the fusion partner may comprise oligo-histidine or other amino acids which bind to cobalt or nickel ions. It may also be an epitope for a monoclonal antibody such as a Myc epitope.
  • variant polypeptides or polypeptide fragments, or fusions of these are typically ones which give rise to neutralizing antibodies against N. meningitidis.
  • the invention also includes, therefore, a method of making an antigen as described above, and antigens obtainable or obtained by the method.
  • the polynucleotides of the invention may be cloned into vectors, such as expression vectors, as is well known on the art.
  • vectors may be present in host cells, such as bacterial, yeast, mammalian and insect host cells.
  • the antigens of the invention may readily be expressed from polynucleotides in a suitable host cell, and isolated therefrom for use in a vaccine.
  • Typical expression systems include the commercially available pET expression vector series and E. coli host cells such as BL21.
  • the polypeptides expressed may be purified by any method known in the art.
  • the antigen is fused to a fusion partner that binds to an affinity column as discussed above, and the fusion is purified using the affinity column (eg such as a nickel or cobalt affinity column).
  • the antigen or a polynucleotide encoding the antigen is particularly suited for use as in a vaccine.
  • the antigen is purified from the host cell it is produced in (or if produced by peptide synthesis purified from any contaminants of the synthesis).
  • the antigen contains less that 5% of contaminating material, preferably less than 2%, 1%, 0.5%, 0.1%, 0.01%, before it is formulated for use in a vaccine.
  • the antigen desirably is substantially pyrogen free.
  • the invention further includes a vaccine comprising the antigen, and method for making a vaccine comprising combining the antigen with a suitable carrier, such as phosphate buffered saline.
  • a suitable carrier such as phosphate buffered saline.
  • an antigen of the invention Whilst it is possible for an antigen of the invention to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers.
  • the carrier(s) must be "acceptable” in the sense of being compatible with the antigen of the invention and not deleterious to the recipients thereof. Typically, the carriers will be water or saline which will be sterile and pyrogen free.
  • the vaccine may also conveniently include an adjuvant. Active immunisation of the patient is preferred.
  • one or more antigens are prepared in an immunogenic formulation containing suitable adjuvants and carriers and administered to the patient in known ways.
  • suitable adjuvants include Freund's complete or incomplete adjuvant, muramyl dipeptide, the "Iscoms" of EP 109 942, EP 180 564 and EP 231 039, aluminium hydroxide, saponin, DEAE-dextran, neutral oils (such as miglyol), vegetable oils (such as arachis oil), liposomes, Pluronic polyols or the Ribi adjuvant system (see, for example GB-A-2 189 141). "Pluronic" is a Registered Trade Mark.
  • the patient to be immunised is a patient requiring to be protected from infection with the microorganism.
  • the invention also includes a pharmaceutical composition comprising a polypeptide of the invention or variant or fragment thereof, or fusion of these, or a polynucleotide of the invention or a variant or fragment thereof or fusion of these, and a pharmaceutically acceptable carrier as discussed above.
  • the aforementioned antigens of the invention may be administered by any conventional method including oral and parenteral (eg subcutaneous or intramuscular) injection.
  • the treatment may consist of a single dose or a plurality of doses over a period of time.
  • the vaccine of the invention may be useful in the fields of human medicine and veterinary medicine.
  • the vaccines of the invention when containing an appropriate antigen or polynucleotide encoding an antigen, are useful in man but also in, for example, cows, sheep, pigs, horses, dogs and cats, and in poultry such as chickens, turkeys, ducks and geese.
  • the invention also includes a method of vaccinating an individual against a microorganism, the method comprising administering to the individual an antigen (or polynucleotide encoding an antigen) or vaccine as described above.
  • the invention also includes the use of the antigen (or polynucleotide encoding an antigen) as described above in the manufacture of a vaccine for vaccinating an individual.
  • the antigen of the invention may be used as the sole antigen in a vaccine or it may be used in combination with other antigens whether directed at the same or different disease microorganisms.
  • the antigen obtained which is reactive against NmB may be combined with components used in vaccines for the A and/or C serogroups. It may also conveniently be combined antigenic components which provide protection against Haemophilus and/or Streptococcus pneumoniae.
  • the additional antigenic components may be polypeptides or they may be other antigenic components such as a polysaccharide. Polysaccharides may also be used to enhance the immune response (see, for example, Makela et al (2002) Expert Rev. Vaccines 1, 399-410).
  • the antigen is the polypeptide encoded by any of the genes as described above (and in the Examples), or a variant or fragment or fusion as described above (or a polynucleotide encoding said antigen), and that the disease to be vaccinated against is Neisseria meningitidis infection (meningococcal disease).
  • GSI GSI is described in more detail in PCT/GB2005/005441 (published as WO 2005/060995 on " 7 July 2005).
  • TspA is a surface antigen which elicits strong CD4+ T cell responses and is recognized by sera from patients (Kizil et al (1999) Infect Immun. 67, 3533-41).
  • NMB0338 is a gene of previously unknown function which encodes a polypeptide that is predicted to contain two transmembrane domains, and is located at the cell surface.
  • the amino acid sequence encoded by NMB0338 is: MERNGVFGKIVGNRILRMSSEHAAASYPKPCKSFKLAQSWFRVRSCLGGVFIYGA NMKLIYTVIKIIILLLFLLLAVINTDAVTFSYLPGQKFDLPLIVYLFGAFWGII FGMFALFGRLLSLRGENGRLRAEVKKNARLTGKELTAPPAQNAPESTKQP
  • JVwB JVwB for GSI aside from the public health imperative: a) the bacterium is genetically tractable; b) killing of the bacterium by effector immune mechanism is straightforward to assay; c) the genome sequences are available for three isolates of different serogroups and clonal lineages (IV-A, ET-5, and ET-37 for serogroups A, B 5 and C, respectively); and d) well-characterised clinical resources are available for this work.
  • GSI has two potential limitations. First, targets of bactericidal antibodies may be essential. This is unlikely as all known targets of bactericidal antibodies in JVmB are non-essential, and no currently licensed bacterial vaccine targets an essential gene product. Second, sera will contain antibodies to multiple antigens, and, loss of a single antigen may not affect the survival of mutants. We have already shown that even during selection with sera raised against the homologus strain, relevant antigens were still identified using appropriate dilutions of sera.
  • GSI GSI will rapidly pinpoint the subset of surface proteins that elicit bactericidal activity, allowing more detailed analysis of a smaller number of candidates.
  • pre- and post-immunisation samples provided by the Meningococcal Reference Laboratory
  • OMVs outer membrane vesicle
  • Murine 1 Defined antigenic exposure.
  • GSI is a high-throughput analysis performed using simple, available techniques. Antigens which elicit bactericidal antibodies in humans and which mediate killing of multiple strains can be identified rapidly as GSI is flexible with respect to the bacterial strain and sera used. Mutants selected using human sera are analysed in the same way as those selected by murine sera.
  • Proteins which are targets of bactericidal antibodies that are recognised by sera from convalescent patients and vaccines are expressed in E. coli using commercially available vectors.
  • the corresponding open reading frames are amplified by PCR from MC58, and ligated into vectors such as pCR Topo CT or pBAD/His, to allow protein expression under the control of a T7 or arabinose- inducible promoter, respectively.
  • Purification of the recombinant proteins from total cellular protein is performed via the His Tag fused to the C terminus of the protein on a Nickel or Cobalt column.
  • SBAs will be performed against MC58 (the homologous strain), and the sequenced serogroup A and C strains with the rabbit immune serum.
  • the assay will be performed in triplicate on at least two occasions. SBAs of >8 will be considered significant. The results provide evidence of whether the protein candidates can elicit bactericidal antibodies as recombinant proteins.
  • mice are immunised on days 0 and 21, and on day 28 receive live bacterial challenge of 10 or 10 7 CFU of MC58 intraperitoneally in iron dextran (as the supplemental iron source).
  • the model is similar to that described for evaluation of the protective efficacy of immunisation with Tbps Danve et al (1993) Vaccine 11, 1214-1220.
  • Non-immunised animals develop bacteraemia within 4 hours of infection, and show signs of systemic illness by 24 hours.
  • PorA is an outer membrane protein that elicits bactericidal antibodies, but which is not a lead vaccine candidate because of extensive antigenic variation (Bart et al (1999) Infect Immun. 67, 3832- 3846.
  • mice Six week old, BALB/c mice (group size, 35 animals) receive 25 ⁇ g of recombinant protein with Freund's incomplete adjuvant subcutaneously on days day 0 and 21, then are challenged with 10 6 (15 animals) or 10 7 (15 animals) CFU of MC58 intraperitoneally on day 28. Two challenge doses are used to examine the vaccine efficacy at a high and low challenge dose; sera are obtained on day 28 from the remaining five animals in each group, and from five animals before the first immunisation and stored at -7O 0 C for further immunological assays. Animals in control groups receive either i) adjuvant alone, ii) recombinant refolded PorA, and iii) a live, attenuated Nm strain.
  • bacteraemia is maximal at this time. The results are analysed using a two-tailed Student-T test to determine if there is a significant reduction in bacteraemia in vaccinated animals.
  • mutants were constructed by in vitro mutagenesis. Genomic DNA from N. meningitidis was subjected to mutagenesis with a Tn5 derivative containing a marker encoding resistance to kanamycin, and an origin of replication which is functional in E. coli. These elements are bound by composite Tn5 ends. Transposition reactions were carried out with a hyperactive variant of Tn5 and the DNA repaired with T4 DNA polymerase and ligase in the presence of ATP and nucleotides. The repaired DNA was used to transform N. meningitidis to kaiiamycin resistance. Southern analysis confirmed that each mutant contained a single insertion of the transposon only.
  • SBAs Serum bactericidal assays
  • Bacteria were grown overnight on solid media (brain heart infusion media with Levanthals supplement) and then re-streaked to solid media for four hours on the morning of experiments. After this time, bacteria were harvested into phosphate buffered saline and enumerated. SBAs were performed in a 1 ml volume, containing a complement source (baby rabbit or human) and approximately 10 5 colony forming units. The bacteria were collected at the end of the incubation and plated to solid media to recover surviving bacteria.
  • Genomic DNA will be recovered from mutants of interest by standard methods and digested with PvuTL, EcoRV, and Dral for three hours, then purified by phenol extraction. The DNA will then be self-ligated in a 100 microlitre volume overnight at 16 0 C in the presence of T4 DNA ligase, precipitated, then used to transform E. coli to kanamycin resistance by electroporation.
  • GSI has been used to screen a library of approximately 40,000 insertional mutants of MC58.
  • the library was constructed by in vitro Tn5 mutagenesis, using a transposon harbouring the origin of replication from pACYC184.
  • MC58 was chosen as it is a sero group B isolate of JV. meningitidis, and the complete genome sequence of this strain is known.
  • the library is always screened in parallel with the wild-type strain as a control, and the number of colonies recovered from the library and the wild-type is shown. Selection with murine sera
  • the screen identified several mutants with enhanced resistance to serum killing: This was confirmed by isolating individual mutants, reconstructing the mutation in the original genetic background, and re-testing the individual mutants for their susceptibility to complement mediated lysis against the wild-tye.
  • the transposon insertions areinthefollowing gene:
  • polypeptide indicates that it is predicted to have two membrane spanning domains, from residues 54 to 70 and 88 to 107.
  • fragments from the regions 1 to 53, and 108 to the end (C-terminal) may be particularly useful as immunogens.
  • NMB2056 Protein sequence MNGKYYYGTGRRKSSVARVFLIKGTGQIIVNGRPVDEFFARETSRMWRQPLVLTENAES FDIKVNVVGGGETGQSGAIRHGITRALIDFDAALKPALSQAGFVTRDAR ⁇ VERKKPGLRK
  • NMB1710 Glutamate dehydrogenase(gdhA) DNA sequence ATGACTGACCTGAACACCCTGTTTGCCAACCTCAAACAACGCAATCCCAATCAGGAGCCG TTCCATCAGGCGGTTGAAGAAGTCTTCATGAGTCTCGATCCGTTTTTGGCA ⁇ AAAATCCG AAATACACCC ⁇ GCAAAGCCTGCTGGAACGCATCGTCGAACCCGAACGCGTCGTGATGTTC CGCGTAACCTGGCAGGACGATAAAGGGCAAGTCCAAGTCAACCGGGGCTACCGCGTGCAA ATGAGTTCCGCCATCGGTCCTTACAAAGGCGGCCTGCGCTTCCATCCGACCGTCGATTTG GGCGTATTG ⁇ A ⁇ TTCCTCGCTTTTG ⁇ ACAAGTGTTCA ⁇ AA ⁇ CGCCTTGACCACCCTGCCT ATGGGCGGCGGCAAAGGCGGTTCCGACTTCGACCCCAAAGGCAAATCCGATGCCGAAGTA ATGCGCTTCTGCCAAGCCTTTATGACCGAACTCTACCGCCACATCGGCGG
  • NMB1333 Amino acid sequence MRYKPLLLALMLVFSTPAVAAHDAAHNRSAEVKKQTKNKKEQPEAAEGKKEKGKNGAVKD KKTGGKEAAKEGKESKKTAKNRKEAEKEATSRQSARKGREGDKKSKAEHKKAHGKPVSGS KEKNAKTQPENKQGKKEAKGQGNPRKGGK ⁇ EKDTVSANKKVRSDKNGKAVKQDKKYREEK N ⁇ KTDSDELKAAVAAATNDVENKKALLKQSEGMLLHVSNSLKQLQEERIRQERIRQARGN LASVNRKQREAWDKFQKLNTELNRLKTEVAATKAQISRFVSGNYKNSQPNAVALFLKNAE PGQKNRFLRYTRYVNASNREWKDLEKQQKALAVQEQKINNELARLKKIQANVQSLLKKQ GVTDAAEQTESRRQNAKIAKDARKLLEQKGNEQQLNKLLSNLEKKKAEHR
  • NMB1036 Amino acid sequence MTAQTLYDKLWNSHWREEEDGTVLLYIDRHLVHEVTSPQAFEGLKMAGRKLWRIDSVVS TADHNTPTGDWDKGIQDPISKLQVDTLDKNIKEFGALAYFPFMDKGQGIVHVMGPEQGAT LPGMTWCGDSHTSTHGAFGALAHGIGTSEVEHTM ⁇ TQCIT ⁇ KKSKSMLISVDGKLKAGV TAKDVALYIIGQIGTAGGTGYAIEFGGEAIRSLSMESRMTLCNMAIEAGARSGMVAVDQT TIDYVKDKPFAPEGEAWDKAVEYWRTLVSDEGAVFDKEYRFNAEDIEPQVTWGTSPEMVL DISSKVPNPAEETDPVKRSGMERALEYMGLEAGTPLNEIPVDIVFIGSCTNSRIEDLREA AAIAKDRKKAANVQRVLIVPGSGLVKEQAEKEGLDKIFIEAGFEWREPGCSMCLAMNADR LTPGQRC
  • NMB1176 Nucleic acid sequence ATGAAAGACAAGCACGATTCTTCCGCCATGCGGCTGGACAAATGGCTTTGGGCGGCACGT TTTTTCA ⁇ GACCCGTTCCCTTGCGCAAAAGCACATCGAACTGGGTAGGGTTCAAGTAAAC GGCTCGAAGGTCAAAAACAGTAAAACCATAGACATCGGCGATATTATCGACCTGACGCTC AATTCCCTTCCCTATAAAATCAAGGTTA ⁇ AGGTTTGAACCACCAACGCCGCCCGGCATCC GAGGCGCGGCTTCTGTATGAAGAGGACGCGAAAACGGCAACATTGAGGGAAGAGCGCAAA CAGCTCGACCAATTCAGCCGCATCACTTCCGCCTATCCCGACGGCAGACCGACCAAGCGC GACCGCCGCCAACTGGACAGGCTGAAAAAAAAGGAGACTGGTAA
  • CAGGCTTTATCCGAAGCAGAAAAAGCGCAAGGCAAGATTTTGATGTGCTGCACCACTGCG CAA ⁇ GCG ⁇ TATCAACATCAACATCCCCGGCTACAAAGCCGATGCCCTACCCGTCCGCACC CTGCCCGCACGCATCGAAAGTATTATTTTCAA ⁇ C ⁇ CGATGTCGCCCTCCTGAA ⁇ CTTGCC CTGCCCAAAGCCCCGCCGTTTGCCTTCTACGCCGGGCAATACATTGATTTACTGCTGCCG GGCA ⁇ CGTCAGCCGCAGCTACTCCATCGCCAATTTACCCGACCAAGAAGGCATTTTGGAA CTGCACATCCGCAGGCACGAAAACGGTGTCTGCTCGGAAATGATTTTCGGCAGCGAACCC

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Epidemiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Communicable Diseases (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Oncology (AREA)
  • Inorganic Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Various polypeptides, or a variant or fragment thereof or a fusion of these are described which are useful in a vaccine. The polypeptide may be a polypeptide comprising the amino acid sequence selected from any one of SEQ ID Nos (2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68); or a fragment or variant thereof or a fusion of such fragment or variant, and is useful in a vaccine against Neisseira meningitidis.

Description

VACCINES AND THEIR USE
The present invention relates to vaccines and their use, and in particular to vaccines for meningococcal disease.
The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge. The documents listed in the specification are hereby incorporated by reference.
Microbial infections remain a serious risk to human and animal health, particularly in light of the fact that many pathogenic microorganisms, particularly bacteria, are or may become resistant to anti-microbial agents such as antibiotics.
Vaccination provides an alternative approach to combating microbial infections, but it is often difficult to identify suitable imrnunogens for use in vaccines which are safe and which are effective against a range of different isolates of a pathogenic microorganism, particular a genetically diverse microorganism. Although it is possible to develop vaccines which use as the immunogen substantially intact microorganisms, such as live attenuated bacteria which typically contain one or mutations in a virulence-deterrnining gene, not all microorganisms are amenable to this approach, and it is not always desirable to adopt this approach for a particular microorganism where safety cannot always be guaranteed. Also, some microorganisms express molecules which mimic host proteins, and these are undesirable in a vaccine.
A particular group of microorganisms for which it is important to develop further vaccines is Neisseria meningitidis which causes meningococcal disease, a life threatening infection which in the Europe, North America, developing countries and elsewhere remains an important cause of childhood mortality despite the introduction of the conjugate serogroup C polysaccharide vaccine. This is because infections caused by serogroup B strains (NmB), which express an cc2-8 linked polysialic acid capsule, are still prevalent. The term "serogroup" in relation to N. meningitidis refers to the polysaccharide capsule expressed on the bacterium. The common serogroup in the UK causing disease is B, while in Africa it is A. Meningococcal septicaemia continues to carry a high case fatality rate; and survivors are often left with major psychological and/or physical disability. After a non-specific prodromal illness, meningococcal septicaemia can present as a fulminant disease that is refractory to appropriate anti-microbial therapy and full supportive measures. Therefore, the best approach to combating the public health menace of meningococcal disease is through prophylactic vaccination.
The non-specific early clinical signs and fulminant course of meningococcal infection mean that therapy is often ineffective. Therefore vaccination is considered the most effective, strategy to diminish the global disease burden caused by this pathogen (Feavers (2000) ABC of meningococcal diversity. Nature 404, 451-2). Existing vaccines to prevent serogroup A, C, W135, and Y TV. meningitidis infections are based on the polysaccharide capsule located on the surface of bacterium (Anderson et al (1994) Safety and immunogenicity of meningococcal A and C polysaccharide conjugate vaccine in adults. Infect Immun. 62, 3391-33955; Leach et al (1997) Induction of immunologic memory in Gambian children by vaccination in infancy with a group A plus group C meningococcal polysaccharide-protein conjugate vaccine. J Infect Dis. 175, 200-4; Lieberman et al (1996). Safety and immunogenicity of a serogroups A/C Neisseria meningitidis oligosaccharide-protein conjugate vaccine in young children. A randomized controlled trial. J. American Med. Assoc. 275, 1499-1503). Progress toward a vaccine against serogroup B infections has been more difficult as its capsule, a homopolymer of α2-8 linked sialic acid, is a relatively poor immunogen in humans. This is because it shares epitopes expressed on a human cell adhesion molecule, N-CAMl (Finne et al (1983) Antigenic similarities between brain components and bacteria causing meningitis. Implications for vaccine development and pathogenesis. Lancet 2, 355-357). Indeed, generating immune responses against the serogroup B capsule might actually prove harmful. Thus, there remains a need for new vaccines to prevent serogroup B N. meningitidis infections.
The most validated immunologic correlate of protection against meningococcal disease is the serum bactericidal assay (SBA). The SBA evaluates the ability of antibodies (usually IgG2a subclass) in serum to mediate complement deposition on the bacterial cell surface, assembly of the membrane attack complex, and bacterial lysis. In the SBA, a known number of bacteria are exposed serial dilutions of the sera with a defined complement source. The number of surviving bacteria is determined, and the SBA is defined as the reciprocal of the highest dilution of serum that mediates 50% killing. The SBA is predictive of protection against serogroup C infections, and has been widely used as a surrogate for immunity against NmB infections. Importantly the SBA is a ready marker of immunity for the pre-clinical assessment of vaccines, and provides a suitable endpoint in clinical trials.
Most efforts at TVmB vaccine development are directed toward defining effective protein sύbunits. There has been a major investment in 'Reverse vaccinology', in which genome sequences are interrogated for potentially surface expressed proteins which are expressed as heterologous antigens and tested for their ability to generate meaningful responses in animals. However, this approach is limited by 1) the computer algorithms for predicting surface expressed antigens, 2) failure to express many of potential immunogens, and 3) the total reliance on murine immune responses.
The key to a successful vaccine is to define antigen(s) that elicit protection against a broad range of disease isolates irrespective of serogroup or clonal group. A genetic screening method (which we have termed Genetic Screening for Immunogens or GSI) was used to isolate antigens that are conserved across the genetic diversity of microbial strains and this is exemplified in relation to meningococcal strains. This was done by identifying microbial antigens, such as K meningitidis antigens, by GSI as described in more detail below; and validated by assessing the function of the immune response elicited by the recombinant antigens and by evaluating the protective efficacy of antigens (see Examples and see PCT/GB2004/005441 (published as WO 2005/060995 on 7 July 2005) incorporated herein by reference). In essence, the GSI method relates to a method for identifying a polypeptide of a microorganism which polypeptide is associated with an immune response in an animal which has been subjected to the microorganism, the method comprising the steps of (1) providing a plurality of different mutants of the microorganism; (2) contacting the plurality of mutant microorganisms with antibodies from an animal which has raised an immune response to the microorganism or a part thereof, under conditions whereby if the antibodies bind to the mutant microorganism the mutant microorganism is killed; (3) selecting surviving mutant microorganisms from step (2); (4) identifying the gene containing the mutation in any surviving mutant microorganism; and (5) identifying the polypeptide encoded by the gene. It will be appreciated that by the way in which the polypeptides have been identified, they are highly relevant as antigenic polypeptides.
As described in more detail in the Examples, particular genes identified by the GSI method are the NBM0341 (TspA), NMB0338, NMB1345, NMB0738, NBM0792 (NadC family), NMB0279, NMB2050, NMB1335 (CreA), NMB2035, NMB1351 (Fmu and Fmv), NMB1574 (HvC), NMB1298 (rsuA), NMB1856 (LysR family), NMBOl 19, NMB1705 (rfak), NMB2065 (HemK), NMB0339, NMB0401 (putA), NMB 1467 (PPX), NMB2056, NMB0808, NMB0774 (upp), NMA0078, NMB0337 (branched-chain amino acid transferase), NMB0191 (ParA family), NMB1710 (glutamate dehydrogenase (gdhA), NMB0062 (rfbA-1), NMB1583 (MsB), NMB0377, NMB0264, NMB1333, NMB1036, NMBl 176, NMB1359 and NMB1138 genes of Neisseria meningitidis. The genome sequence for N. meningitidis is available, for example from The Institute of Genome Research (TIGR); www.tigr.org.
Although these genes form part of the genome that has been sequenced, as far as the inventors are aware, they have not been isolated, the polypeptides they encode have not been produced (and have not been isolated), and there is no indication that the polypeptides they encode may be useful as a component of a vaccine.
Thus, the invention includes the isolated genes as above and in the Examples and variants and fragments and fusions of such variants and fragments, and includes the polypeptides that the genes encode as described above, along with variants and fragment thereof, and fusions of such fragments and variants. Variants, fragments and fusions are described hi more detail below. Preferably, the variants, fragments and fusions of the given genes above are ones which encode a polypeptide which gives rise to neutralizing antibodies against N. meningitidis. Similarly, preferably, the variants, fragments and fusions of the polypeptide whose sequence is given above are ones which gives rise to neutralizing antibodies against N. meningitidis. The neutralising antibodies may be produced in any animal with an immune system, for example a rat, mouse or rabbit. The invention also includes isolated polynucleotides encoding the polypeptides whose sequences are given in the Example (preferably the isolated coding region) or encoding the variants, fragments or fusions. The invention also includes expression vectors comprising such polynucleotides and host cells comprising such polynucleotides and vectors (as is described in more detail below). The polypeptides described in the Examples are antigens identified by the method of the invention.
Molecular biological methods for use in the practice of the method of the invention are well known in the art, for example from Sambrook & Russell (2001) Molecular Cloning, a laboratory manual, third edition, Cold Spring Harbor laboratory Press, Cold Spring Harbor, New York, incorporated herein by reference.
Variants of the gene may be made, for example by identifying related genes in other microorganisms or in other strains of the microorganism, and cloning, isolating or synthesizing the gene. Typically, variants of the gene are ones which have at least 70% sequence identity, more preferably at least 85% sequence identity, most preferably at least 95% sequence identity with the genes as given above. Of course, replacements, deletions and insertions may be tolerated. The degree of similarity between one nucleic acid sequence and another can be determined using the GAP program of the University of Wisconsin Computer Group.
Variants of the gene are also ones which hybridise under stringent conditions to the gene. By "stringent" we mean that the gene hybridises to the probe when the gene is immobilised on a membrane and the probe (which, in this case is >200 nucleotides in length) is in solution and the immobilised gene/hybridised probe is washed in 0.1 x SSC at 65°C for 10 min. SSC is 0.15 M NaCl/0.015 M Na citrate.
Fragments of the gene (or the variant gene) may be made which are, for example, 20% or 30% or 40% or 50% or 60% or 70% or 80% or 90% of the total of the gene. Preferred fragments include all or part of the coding sequence. The variant and fragments may be fused to other, unrelated, polynucleotides.
The polynucleotide encodes a polypeptide which is immunogenic and is reactive with the antibodies from an animal which has been subjected to the microorganism from which the gene was identified.
The antigen may be the polypeptide as encoded by the gene identified above, and the sequence of the polypeptide may readily be deduced from the gene sequence. In further embodiments, the antigen may be a fragment of the identified polypeptide or may be a variant of the identified polypeptide or may be a fusion of the polypeptide or fragment or variant.
Thus, a particular aspect of the invention provides a polypeptide comprising the amino acid sequence selected from any one of SEQ ID Nos 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68; or a fragment or variant thereof or a fusion of such a fragment or variant. Thus, the invention provides the following isolated proteins, or fragments or variants thereof, or fusion of these: NMB0341, NMB 1583,
NMB1345, NMB0738, NMB0792, NMB0279, NMB2050, NMB1335,
NMB2035, NMB1351, NMB1574, NMB1298, NMB1856, NMB0119,
NMB1705, NMB2065, NMB0339, NMB0401, NMB1467, NMB2056, NMB0808, NMB0774, NMA0078, NMB0337, NMB0191, NMB1710, NMB0062, NMB1333, NMB0377, NMB0264, NMB1036, NMBl 176, NMB1359 and NMB 1138 as described below.
Fragments of the identified polypeptide may be made which are, for example, 20% or 30% or 40 % or 50% or 60% or 70% or 80% or 90% of the total of the polypeptide. Typically, fragments are at least 10, 15, 20, 30, 40 , 50, 100 or more amino acids, but less than 500, 400, 300 or 200 amino acids. Variants of the polypeptide may be made. By "variants" we include insertions, deletions and substitutions, either conservative or non-conservative, where such changes do not substantially alter the normal function of the protein. By "conservative substitutions" is intended combinations such as GIy, Ala; VaI, He, Leu; Asp, GIu; Asn, GIn; Ser, Thr; Lys, Arg; and Phe, Tyr. Such variants may be made using the well known methods of protein engineering and site-directed mutagenesis.
A particular class of variants are those encoded by variant genes as discussed above, for example from related microorganisms or other strains of the microorganism. Typically the variant polypeptides have at least 70% sequence identity, more preferably at least 85% sequence identity, most preferably at least 95% sequence identity with the polypeptide identified using the method of the invention.
The percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequence has been aligned optimally. The alignment may alternatively be carried out using the Clustal W program (Thompson et al, (1994) Nucleic Acids Res 22, 4673-80). The parameters used may be as follows:
Fast pairwise alignment parameters: K-tuple(word) size; 1, window size; 5, gap penalty; 3, number of top diagonals; 5. Scoring method: x percent.
Multiple alignment parameters: gap open penalty; 10, gap extension penalty; 0.05. Scoring matrix: BLOSUM.
The fusions may be fusions with any suitable polypeptide. Typically, the polypeptide is one which is able to enhance the immune response to the polypeptide it is fused to. The fusion partner may be a polypeptide that facilitates purification, for example by constituting a binding site for a moiety that can be immobilised in, for example, an affinity chromatography column. Thus, the fusion partner may comprise oligo-histidine or other amino acids which bind to cobalt or nickel ions. It may also be an epitope for a monoclonal antibody such as a Myc epitope.
As discussed above, the variant polypeptides or polypeptide fragments, or fusions of these, are typically ones which give rise to neutralizing antibodies against N. meningitidis.
The invention also includes, therefore, a method of making an antigen as described above, and antigens obtainable or obtained by the method.
The polynucleotides of the invention may be cloned into vectors, such as expression vectors, as is well known on the art. Such vectors maybe present in host cells, such as bacterial, yeast, mammalian and insect host cells. The antigens of the invention may readily be expressed from polynucleotides in a suitable host cell, and isolated therefrom for use in a vaccine.
Typical expression systems include the commercially available pET expression vector series and E. coli host cells such as BL21. The polypeptides expressed may be purified by any method known in the art. Conveniently, the antigen is fused to a fusion partner that binds to an affinity column as discussed above, and the fusion is purified using the affinity column (eg such as a nickel or cobalt affinity column).
It will be appreciated that the antigen or a polynucleotide encoding the antigen (such as a DNA molecule) is particularly suited for use as in a vaccine. In that case, the antigen is purified from the host cell it is produced in (or if produced by peptide synthesis purified from any contaminants of the synthesis). Typically the antigen contains less that 5% of contaminating material, preferably less than 2%, 1%, 0.5%, 0.1%, 0.01%, before it is formulated for use in a vaccine. The antigen desirably is substantially pyrogen free. Thus, the invention further includes a vaccine comprising the antigen, and method for making a vaccine comprising combining the antigen with a suitable carrier, such as phosphate buffered saline. Whilst it is possible for an antigen of the invention to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers. The carrier(s) must be "acceptable" in the sense of being compatible with the antigen of the invention and not deleterious to the recipients thereof. Typically, the carriers will be water or saline which will be sterile and pyrogen free.
The vaccine may also conveniently include an adjuvant. Active immunisation of the patient is preferred. In this approach, one or more antigens are prepared in an immunogenic formulation containing suitable adjuvants and carriers and administered to the patient in known ways. Suitable adjuvants include Freund's complete or incomplete adjuvant, muramyl dipeptide, the "Iscoms" of EP 109 942, EP 180 564 and EP 231 039, aluminium hydroxide, saponin, DEAE-dextran, neutral oils (such as miglyol), vegetable oils (such as arachis oil), liposomes, Pluronic polyols or the Ribi adjuvant system (see, for example GB-A-2 189 141). "Pluronic" is a Registered Trade Mark. The patient to be immunised is a patient requiring to be protected from infection with the microorganism. The invention also includes a pharmaceutical composition comprising a polypeptide of the invention or variant or fragment thereof, or fusion of these, or a polynucleotide of the invention or a variant or fragment thereof or fusion of these, and a pharmaceutically acceptable carrier as discussed above.
The aforementioned antigens of the invention (or polynucleotides encoding such antigens) or a formulation thereof may be administered by any conventional method including oral and parenteral (eg subcutaneous or intramuscular) injection. The treatment may consist of a single dose or a plurality of doses over a period of time.
It will be appreciated that the vaccine of the invention, depending on its antigen component (or polynucleotide), may be useful in the fields of human medicine and veterinary medicine.
Diseases caused by microorganisms are known in many animals, such as domestic animals. The vaccines of the invention, when containing an appropriate antigen or polynucleotide encoding an antigen, are useful in man but also in, for example, cows, sheep, pigs, horses, dogs and cats, and in poultry such as chickens, turkeys, ducks and geese.
Thus, the invention also includes a method of vaccinating an individual against a microorganism, the method comprising administering to the individual an antigen (or polynucleotide encoding an antigen) or vaccine as described above. The invention also includes the use of the antigen (or polynucleotide encoding an antigen) as described above in the manufacture of a vaccine for vaccinating an individual.
The antigen of the invention may be used as the sole antigen in a vaccine or it may be used in combination with other antigens whether directed at the same or different disease microorganisms. In relation to N. meningitidis, the antigen obtained which is reactive against NmB may be combined with components used in vaccines for the A and/or C serogroups. It may also conveniently be combined antigenic components which provide protection against Haemophilus and/or Streptococcus pneumoniae. The additional antigenic components may be polypeptides or they may be other antigenic components such as a polysaccharide. Polysaccharides may also be used to enhance the immune response (see, for example, Makela et al (2002) Expert Rev. Vaccines 1, 399-410).
It is particularly preferred in the above vaccines and methods of vaccination if the antigen is the polypeptide encoded by any of the genes as described above (and in the Examples), or a variant or fragment or fusion as described above (or a polynucleotide encoding said antigen), and that the disease to be vaccinated against is Neisseria meningitidis infection (meningococcal disease).
The invention will now be described in greater detail by reference to the following non-limiting Examples.
Example 1: Genetic screening for immunogens (GSI) in N. meningitidis
The application of GSI in this example involves screening libraries of insertional mutants of N. meningitidis for strains which are less susceptible to killing by bactericidal antibodies. GSI is described in more detail in PCT/GB2005/005441 (published as WO 2005/060995 on"7 July 2005).
We have demonstrated the effectiveness of GSI by screening a library of mutants of the sequenced TVmB isolate, MC58, with sera raised in mice against a capsule minus of the same strain. A total of 40,000 mutants was analysed with sera raised in mice by intraperitoneal immunisation with the homologous strain; the SBA of this sera is around 2,000 against the wild-type strain. Surviving mutants were detected when the library was exposed to serum at a 1:560 dilution (which kills all wild-type bacteria). To establish whether the transposon insertion in the surviving mutants was responsible for the ability to withstand killing, the mutations were backcrossed into the parental strain, and the backcrossed mutants were confirmed as being more resistant to killing than the wild-type in the SBA. The sequence of the gene affected by the transposon was examined by isolating the transposon insertion site by marker rescue. We found that two of the genes affected were TspA and NMB0338. TspA is a surface antigen which elicits strong CD4+ T cell responses and is recognized by sera from patients (Kizil et al (1999) Infect Immun. 67, 3533-41). NMB0338 is a gene of previously unknown function which encodes a polypeptide that is predicted to contain two transmembrane domains, and is located at the cell surface. The amino acid sequence encoded by NMB0338 is: MERNGVFGKIVGNRILRMSSEHAAASYPKPCKSFKLAQSWFRVRSCLGGVFIYGA NMKLIYTVIKIIILLLFLLLAVINTDAVTFSYLPGQKFDLPLIVYLFGAFWGII FGMFALFGRLLSLRGENGRLRAEVKKNARLTGKELTAPPAQNAPESTKQP
There are several practical advantages of using JVwB for GSI aside from the public health imperative: a) the bacterium is genetically tractable; b) killing of the bacterium by effector immune mechanism is straightforward to assay; c) the genome sequences are available for three isolates of different serogroups and clonal lineages (IV-A, ET-5, and ET-37 for serogroups A, B5 and C, respectively); and d) well-characterised clinical resources are available for this work.
GSI has two potential limitations. First, targets of bactericidal antibodies may be essential. This is unlikely as all known targets of bactericidal antibodies in JVmB are non-essential, and no currently licensed bacterial vaccine targets an essential gene product. Second, sera will contain antibodies to multiple antigens, and, loss of a single antigen may not affect the survival of mutants. We have already shown that even during selection with sera raised against the homologus strain, relevant antigens were still identified using appropriate dilutions of sera.
The major advantages of GSI are that 1) the high throughput steps do not involve technically demanding or costly procedures (such as protein expression/purification and immunisation), and 2) human samples can be used in the assay rather than relying solely on animal data. GSI will rapidly pinpoint the subset of surface proteins that elicit bactericidal activity, allowing more detailed analysis of a smaller number of candidates.
1. Identification of targets of bactericidal antibodies using GSI Murine sera raised against heterologous strains, and human sera, are used to identify cross-reactive antigens. The sera are obtained from: i) mice immunised by the systemic route with heterologous strains: the strains will be selected and/or constructed to avoid isolates with the same immunotype and sub-serotype. ii) acute and convalescent sera from patients infected with known isolates of TV. meningitidis (provided by Dr R. Wall, Northwick Park) iii) pre- and post-immunisation samples (provided by the Meningococcal Reference Laboratory) from volunteers receiving defined outer membrane vesicle (OMVs) vaccines derived from the NmB isolate, H44/76.
Each of these sources of sera has specific advantages and disadvantages.
Serum source Advantages Disadvantages
Murine 1) Defined antigenic exposure. 1) Animal source of
2) Use of genetically modified strains to material generate immune response.
3) Naϊve samples available
4) Examine individuals responses
Patient sera 1) Human material 1) Background immunity
2) Known strain exposure 2) Limited material
3) Acute and convalescent sera available
Sera following 1) Human material 1) Background immunity immunisation 2) Defined antigenic exposure 2) Limited material with H4476 3) Pre and post immunisation sera OMVs available
4) Examine individuals responses
a) Sera from animals immunised with heterologous strains (ie the sequenced serogroup A or C strains) are used in GSI to select the library of MC58 mutants. We have shown that immunisation with live, attenuated NmB elicits cross-reactive bactericidal antibody responses against serogroup A and C strains. The antigen absent in mutants with enhanced survival in the face of human sera are identified by marker rescue of the disrupted gene.
b) Mutations are identified that confer resistance against killing by heterologous sera, and it is determined whether the gene product is also a target for killing of the sequenced, serogroup A and C strains, Z2491 and FAMl 8 respectively. The genome databases are inspected for homologues of the genes. If a homologue is present, the transposon insertion is amplified from the MC58 mutant and introduced into the serogroup A and C strains by transformation. The relative survival of the mutant and wild-type strain of each serogroup are compared. Thus, GSI can quickly give information whether the targets of bactericidal activity are conserved and accessible in diverse strains of N. meningitidis, irrespective of serogroup, immunotype and subserotype.
c) Mutants with enhanced survival against sera raised in mice are tested using human sera from either convalescent patients or vaccinees receiving heterologous OMV vaccines (derived from H44/76). This addresses the important question of whether the targets are capable of eliciting bactericidal antibodies in human. With other vaccine approaches, this information is only gained at the late, expensive stage of clinical trials that requires GMP manufacture of vaccine candidates.
The advantages are that GSI is a high-throughput analysis performed using simple, available techniques. Antigens which elicit bactericidal antibodies in humans and which mediate killing of multiple strains can be identified rapidly as GSI is flexible with respect to the bacterial strain and sera used. Mutants selected using human sera are analysed in the same way as those selected by murine sera.
2. Assessment of the antibody response of recombinant GSI antigens
Proteins which are targets of bactericidal antibodies that are recognised by sera from convalescent patients and vaccines are expressed in E. coli using commercially available vectors. The corresponding open reading frames are amplified by PCR from MC58, and ligated into vectors such as pCR Topo CT or pBAD/His, to allow protein expression under the control of a T7 or arabinose- inducible promoter, respectively. Purification of the recombinant proteins from total cellular protein is performed via the His Tag fused to the C terminus of the protein on a Nickel or Cobalt column.
Adult New Zealand White rabbits are immunized on two occasions separated by four weeks by subcutaneous injection with 25 μg of purified protein with Freund's incomplete adjuvant. Sera from animals will be checked prior to immunisation for pre-existing anti-Mn antibodies by whole cell ELISA. Animals which have an initial serum titre of <1:2 are used for immunisation experiments. Post- immunisation serum are obtained two weeks after the second immunisation. To confirm that specific antibodies have been raised, pre- and post-iminunisation serum is tested by i) Western analysis against the purified protein and ii) ELISA using cells from the wild-type and the corresponding mutant (generated by GSI).
SBAs will be performed against MC58 (the homologous strain), and the sequenced serogroup A and C strains with the rabbit immune serum. The assay will be performed in triplicate on at least two occasions. SBAs of >8 will be considered significant. The results provide evidence of whether the protein candidates can elicit bactericidal antibodies as recombinant proteins.
3. Establishing the protective efficacy of GSI antigens
AU the candidates are tested for their ability to protect animals against live bacterial challenge as this allows any aspect of immunity (cellular or humoral) to be assessed in a single assay. We have established a model of active immunisation and protection against live bacterial infection. In this model, adult mice are immunised on days 0 and 21, and on day 28 receive live bacterial challenge of 10 or 107 CFU of MC58 intraperitoneally in iron dextran (as the supplemental iron source). The model is similar to that described for evaluation of the protective efficacy of immunisation with Tbps Danve et al (1993) Vaccine 11, 1214-1220. Non-immunised animals develop bacteraemia within 4 hours of infection, and show signs of systemic illness by 24 hours. We have already been able to demonstrate the protective efficacy of both attenuated Nm strains and a protein antigen against live meningococcal challenge; PorA is an outer membrane protein that elicits bactericidal antibodies, but which is not a lead vaccine candidate because of extensive antigenic variation (Bart et al (1999) Infect Immun. 67, 3832- 3846.
Six week old, BALB/c mice (group size, 35 animals) receive 25 μg of recombinant protein with Freund's incomplete adjuvant subcutaneously on days day 0 and 21, then are challenged with 106 (15 animals) or 107 (15 animals) CFU of MC58 intraperitoneally on day 28. Two challenge doses are used to examine the vaccine efficacy at a high and low challenge dose; sera are obtained on day 28 from the remaining five animals in each group, and from five animals before the first immunisation and stored at -7O0C for further immunological assays. Animals in control groups receive either i) adjuvant alone, ii) recombinant refolded PorA, and iii) a live, attenuated Nm strain. To reduce the overall number of animals in control groups, sets of five candidates will be tested at one time (number of groups = 5 candidates + 3 controls). Survival of animals in the groups is compared by Mann Whitney U Test. With group sizes of 15 mice/dose, the experiments are powered to show a 25% difference in survival between groups.
For vaccines which show significant protection against challenge, a repeat experiment is performed to confirm the finding. Furthermore, to establish that vaccination with a candidate also elicits protection against bacteraemia, levels of bacteraemia are determined during the second experiment; blood is sampled at 22 hr post-infection in immunised and un-immunised animals (bacteraemia is maximal at this time). The results are analysed using a two-tailed Student-T test to determine if there is a significant reduction in bacteraemia in vaccinated animals.
Further materials and methods used
Mutagenesis of Neisseria meningitidis
For work with Neisseria meningitidis, mutants were constructed by in vitro mutagenesis. Genomic DNA from N. meningitidis was subjected to mutagenesis with a Tn5 derivative containing a marker encoding resistance to kanamycin, and an origin of replication which is functional in E. coli. These elements are bound by composite Tn5 ends. Transposition reactions were carried out with a hyperactive variant of Tn5 and the DNA repaired with T4 DNA polymerase and ligase in the presence of ATP and nucleotides. The repaired DNA was used to transform N. meningitidis to kaiiamycin resistance. Southern analysis confirmed that each mutant contained a single insertion of the transposon only.
Serum bactericidal assays (SBAs)
Bacteria were grown overnight on solid media (brain heart infusion media with Levanthals supplement) and then re-streaked to solid media for four hours on the morning of experiments. After this time, bacteria were harvested into phosphate buffered saline and enumerated. SBAs were performed in a 1 ml volume, containing a complement source (baby rabbit or human) and approximately 105 colony forming units. The bacteria were collected at the end of the incubation and plated to solid media to recover surviving bacteria.
Isolating the transposon insertion sites
Genomic DNA will be recovered from mutants of interest by standard methods and digested with PvuTL, EcoRV, and Dral for three hours, then purified by phenol extraction. The DNA will then be self-ligated in a 100 microlitre volume overnight at 160C in the presence of T4 DNA ligase, precipitated, then used to transform E. coli to kanamycin resistance by electroporation.
Example 2: Further screening and results thereof
GSI has been used to screen a library of approximately 40,000 insertional mutants of MC58. The library was constructed by in vitro Tn5 mutagenesis, using a transposon harbouring the origin of replication from pACYC184.
MC58 was chosen as it is a sero group B isolate of JV. meningitidis, and the complete genome sequence of this strain is known.
The library is always screened in parallel with the wild-type strain as a control, and the number of colonies recovered from the library and the wild-type is shown. Selection with murine sera
Initially the library was analysed using sera from animals immunised with the attenuated strain YHl02. Adult mice (Balb/C) received 108 colony forming units intra-peritoneally onthree occasions, and serawas collected 10 days after the final immunisation,
The screen identified several mutants with enhanced resistance to serum killing: This was confirmed by isolating individual mutants, reconstructing the mutation in the original genetic background, and re-testing the individual mutants for their susceptibility to complement mediated lysis against the wild-tye. The transposon insertions areinthefollowing gene:
NMB0341 (TspA) DNA sequence
ATGCCCGCCGGCCGACTGCCCCGCCGATGCCCGATGATGACGAAATTTACAGACTGTACG CGGTCAΆACCGTATTCAGCCGCCAACCCACAGGGGATACATCTTGAAAAACAACAGACAA ATCAAΆCTGATTGCCGCCTCCGTCGCAGTTGCCGCATCCTTTCAGGCACATGCTGGΆCTG GGCGGACTGAATATCCAGTCCAACCTTGACGAACCCTTTTCCGGCAGCATTACCGTAACC GGCGAAGAAGCCAAAGCCCTGCTAGGCGGCGGCAGCGTTACCGTTTCCGAAAAAGGCCTG ACCGCCAAAGTCCACAAGTTGGGCGACAAAGCCGTCATTGCCGTTTCTTCCGAACAGGCA GTCCGCGATCCCGTCCTGGTGTTCCGCATCGGCGCAGGCGCACAGGTACGCGAATACACC GCCATCCTCGATCCTGTCGGCTACTCGCCCΆAAACCAAΆTCTGCACTTTCAGACGGCAAG ACACACCGCAAAΆCCGCTCCGACAGCAGAGTCCCAAGAAΆΆTCAAAACGCCAAΆGCCCTC CGCAAAACCGATAAAAAAGACAGCGCGAACGCAGCCGTCAAACCGGCATACAACGGCΆAΆ ACCCATACCGTCCGCAAAGGCGAAACGGTCAAACAGATTGCCGCCGCCATCCGCCCGAAA CACCTGACGCTCGAΆCAGGTTGCCGATGCGCTGCTGAAGGCAAACCCAΆΆTGTTTCCGCA CACGGCAGACTGCGTGCGGGCAGCGTGCTTCACATTCCGAATCTGAΆCAGGATCAAΆGCG GAACAACCCAAACCGCAAACGGCGAAΆCCCAAAGCCGAΆACCGCATCCATGCCGTCCGAA CCGTCCAAACAGGCAACGGTAGAGAAACCGGTTGAAAAACCTGAAGCAAAAGTTGCCGCG CCCGAAGCAAAAGCGGAΆΆAACCGGCCGTTCGACCCGAACCTGTACCCGCTGCAAΆTACT GCCGCATCGGAAACCGCTGCCGAATCCGCCCCCCAAGAAGCCGCCGCTTCTGCCATCGAC ACGCCGACCGACGAAACCGGTAΆCGCCGTTTCCGAACCTGTCGAACAGGTTTCTGCCGAΆ GAΆGAAΆCCGAΆAGCGGACTGTTTGACGGTCTGTTCGGCGGTTCGTACACCTTGCTGCTT GCCGGCGGAGGCGCGGCATTAATCGCCCTGCTGCTGCTTTTGCGCCTTGCCCAATCCAAA CGCGCGCGCCGTACCGAAGAATCCGTCCCTGAGGAAGAGCCTGACCTTGACGACGCGGCA GACGACGGCATAGAAATCACCTTTGCCGAAGTCGAAACTCCGGCAΆCGCCCGAΆCCCGCT CCGAAAAΆCGATGTAAACGACACACTTGCCTTAGATGGGGAATCTGAAGAAGAGTTATCG GCAAAACAAACGTTCGATGTCGAAACCGATACGCCTTCCAACCGCATCGACTTGGATTTC GACAGCCTGGCAGCCGCGCAAAACGGCATTTTATCCGGCGCACTTACGCAGGATGAAGAA ACCCAAAAACGCGCGGATGCCGATTGGAACGCCATCGAATCCACAGACAGCGTGTACGAG CCCGAGACCTTCAACCCGTACAACCCTGTCGAAATCGTCATCGACACGCCCGAΆCCGGAΆ TCTGTCGCCCAAACTGCCGAAAACAAACCGGAAACCGTCGATACCGATTTCTCCGACAΆC CTGCCCTCAAACAACCATATCGGCACAGAAGAAACAGCTTCCGCAAAACCTGCCTCACCC TCCGGACTGGCAGGCTTCCTGAAGGCTTCCTCGCCCGAAACCATCTTGGAAAAAACAGTT GCCGAAGTCCAAACACCGGAAGAGTTGCACGATTTCCTGAAAGTGTACGAΆACCGATGCC GTCGCGGAAΆCTGCGCCTGAΆΆCGCCCGATTTCAACGCCGCCGCAGACGATTTGTCCGCA TTGCTTCAACCTGCCGAAGCACCGTCCGTTGAGGAAAATATAACGGAAACCGTTGCCGAA
ACACCCGACTTCAΆCGCCACCGCAGACGATTTGTCCGCATTACTTCAACCTTCTAAΆGTA CCTGCCGTTGAGGAAAATGCAGCGGAΆACCGTTGCCGATGATTTGTCCGCACTGTTGCAA CCTGCTGAAGCACCGGCCGTTGAGGAAAATGTAACGGAAACCGTTGCCGAAACACCCGAT TTCAΆCGCCACCGCAGACGATTTGTCCGCATTACTTCAΆCCTTCTGAΆGCACCTGCCGTT GAGGAΆAATGCAGCGGAΆACCGTTGCCGATGATTTGTCCGCACTGTTGCAACCTGCTGAA GCACCGGCCGTTGAGGAAAATGCAGCGGAAATCACTTTGGAAΆCGCCTGATTCCAACACC TCTGΆGGCAGACGCTTTGCCCGACTTCCTGAAAGACGGCGAGGAGGAAACGGTAGATTGG AGCATCTACCTCTCGGAAGAAAΆTATCCCAAATAATGCAGATACCAGTTTCCCTTCGGAA TCTGTAGGTTCTGΆCGCGCCTTCCGΆAGCGAAΆTACGACCTTGCCGAAΆTGTATCTCGAA ATCGGCGACCGCGATGCCGCTGCCGΆGACAGTGCAGAAATTGCTGGAAGAΆGCGGAAGGC GACGTACTCAAACGTGCCCAAGCATTGGCGCAGGAATTGGGTATTTGA
NBM0341 Protein sequence MPAGRLPRRCPMMTKFTDCTRSNRIQPPTHRGYILKNNRQIKLIAASVAVAASFQAHAGL GGLNIQSNLDEPFSGSITVTGEEAKALLGGGSVTVSEKGLTAKVHKLGDKAVIAVSSEQA VRDPVLVFRIGAGAQVREYTAILDPVGYSPKTKSALSDGKTHRKTAPTAESOENQNAKAL RKTDKKDSANAAVKPAYNGKTHTVRKGETVKQIAAAIRPKHLTLEQVADALLKANPNVSA
HGRLRAGSVLHiPNLNRiKAEQPKPQTAKPKAETASMPSΞPSKQATVEKPVEKPEAKVAA PEAKAEKPAVRPEPVPAANTAASETAAESAPQEAAASAIDTPTDETGNAVSEPVEQVSAE EETESGLFDGLFGGSYTLLLAGGGAALIALLLLLRLAQSKRARRTEESVPEEEPDLDDAA DDGIEITFAEVETPATPEPAPKNDVNDTLALDGESEEELSAKQTFDVETDTPSNRIDLDF DSLAΆΆQNGILSGALTQDEETQKRADADWNAIESTDSVYEPETFNPYNPVEIVIDTPEPE SVAQTAENKPETVDTDFSDNLPSNNHIGTEETASAKPASPSGLAGFLKASSPETILEKTV AEVQTPEELHDFLKVYETDAVAETAPETPDFNAAADDLSALLQPAEAPSVEENITETVAE TPDFNATADDLSALLQPSKVPAVEENAAETVADDLSALLQPAEAPAVEENVTETVAETPD FNATADDLSALLQPSEAPAVEENAAETVADDLSALLQPAEAPAVEENAΆEITLETPDSNT SEADALPDFLKDGEEETVDWSIYLSEΞNIPNNADTSFPSESVGSDAPSEAKYDLAEMYLE IGDRDAAAETVQKLLEEAEGDVLKRAQALAQELGI
NMB0338 DNA sequence
ATGGAAAGGAACGGTGTATTTGGTAAAATTGTCGGCAATCGCATACTCCGTATGTCGTCC GAACACGCTGCCGCATCCTATCCGAAACCGTGCAAATCGTTTAAACTAGCGCAATCTTGG TTCAGAGTGCGAΆGCTGTCTGGGCGGCGTTTTTATTTACGGAGCAAΆCATGAΆACTTATC TATACCGTCATCAAAATCATTATCCTGCTGCTCTTCCTGCTGCTTGCCGTCATTAATACG GATGCCGTTACCTTTTCCTACCTGCCGGGGCAAAAATTCGATTTGCCGCTGATTGTCGTA TTGTTCGGCGCATTTGTAGTCGGTATTATTTTTGGAATGTTTGCCTTGTTCGGACGGTTG TTGTCGTTACGTGGCGAGAACGGCAGGTTGCGTGCCGAAGTAΆΆGAΆΆAATGCGCGTTTG ACGGGGAAGGAGCTGACCGCACCACCGGCGCAAAATGCGCCCGAATCTACCAAACAGCCT TAA
NMB0338 Protein sequence
MERNGVFGKIVGNRILRMSSEHAAASYPKPCKS FKLAQSWFRVRSCLGGVFIYGANMKLI YTVIKIIILLLFLLLAVINTDAVTFSYLPGQKFDLPLIWLFGAFVVGIIFGMFALFGRL LSLRGENGRLRAEVKKNARLTGKELTAPPAQNAPESTKQP
Analysis of the polypeptide indicates that it is predicted to have two membrane spanning domains, from residues 54 to 70 and 88 to 107. Thus, fragments from the regions 1 to 53, and 108 to the end (C-terminal) may be particularly useful as immunogens.
NMB 1345 DNA sequence
ATGAAAAAACCTTTGATTTCGGTTGCGGCAGCATTGCTCGGCGTTGCTTTGGGCACGCCT TATTATTTGGGTGTCAAAGCCGAAGAAAGCTTGACGCAGCAGCAAAAAATATTGCAGGAA ACGGGCTTCTTGACCGTCGAATCGCACCAATATGAGCGCGGCTGGTTTACCTCTATGGAA ACGACGGTCATCCGTCTGAAACCCGAGTTGCTGAATAATGCCCGAAAATACCTGCCGGAT AACCTGAAAACAGTGTTGGAACAGCCGGTTACGCTGGTTAACCATATCACGCACGGCCCT
TTCGCCGGCGGATTCGGCACGCAGGCGTACATTGAAACCGAGTTCAAATACGCGCCTGAA ACGGAAAAAGTTCTGGAACGCTTTTTTGGAAAACAAGTCCCGGCTTCCCTTGCCAATACC GTTTATTTTAACGGCAGCGGTAAAATGGAAGTCAGTGTTCCCGCCTTCGATTATGAΆGAG CTGTCGGGCΆTCAGGCTGCACTGGGAAGGCCTGACGGGAGAAACGGTTTATCAAAΆAGGT TTCAAAAGCTACCGGAACGGCTATGATGCCCCCTTGTTTΆAAATCAAGCTGGCAGACAAA GGCGATGCCGCGTTTGAAAAAGTGCATTTCGATTCGGAAACTTCAGACGGCATCAATCCG CTTGCTTTGGGCAGCAGCAATCTGACCTTGGAAAAATTCTCCCTAGAATGGAAAGAGGGT GTCGATTACAACGTCAAGTTAAΆCGAACTGGTCAATCTTGTTACCGATTTGCAGATTGGC GCGTTTATCAATCCCAACGGCAGCATCGCACCTTCCAAAATCGAAGTCGGCAAACTGGCT TTTTCAACCΆAGACCGGGGAATCAGGCGCGTTTATCAACAGTGAAGGGCAGTTCCGTTTC GATACACTGGTGTACGGCGATGAAAAATACGGCCCGCTGGACATCCATATCGCTGCCGAA CACCTCGATGCTTCTGCCTTAACCGTATTGAAACGCAΆGTTTGCACAAATTTCCGCCAAΆ AAAATGACCGAGGAACAΆATCCGCAATGATTTGATTGCCGCCGTCAAAGGAGAGGCTTCC GGACTGTTCACCAACAATCCCGTATTGGACATTAAAACTTTCCGATTCACGCTGCCATCG GGAAAAATCGATGTGGGCGGAAAAATCATGTTTAΆAGACATGAΆGAAGGAAGATTTGAAT CAATTGGGTTTGATGCTGAAGAΆAACCGAAGCCGACATCAGAΆTGAGTATTCCCCAAΆAΆ ATGCTGGAAGACTTGGCGGTCAGTCAAGCAGGCAATATTTTCAGCGTCAATGCCGAAGAT GAGGCGGAAGGCAGGGCAAGTCTTGACGACATCAACGAGACCTTGCGCCTGATGGTGGAC AGTACGGTTCAGAGTATGGCAAGGGAAAAATATCTGACTTTGAACGGCGACCAGATTGAT ACTGCCATTTCTCTGAAAΆΆCAATCAGTTGAAATTGAΆCGGTAAAACGTTGCAAAΆCGAA CCGGAGCCGGATTTTGATGAAGGCGGTATGGTTTCAGAGCCGCAGCAGTAA
NMB 1345 Protein sequence
MKKPLISVAAALLGVALGTPYYLGVKAEESLTQQQKILQETGFLTVESHQYERGWFTSME TTVIRLKPELLNNARKYLPDNLKTVLEQPVTLVNHITHGPFAGGFGTQAYIETEFKYAPE TEKVLERFFGKQVPASLANTVYFNGSGKMEVSVPAFDYEELSGIRLHWEGLTGETVYQKG FKSYRNGYDAPLFKIKLADKGDAAFEKVHFDSETSDGINPLALGSSNLTLEKFSLEWKEG VDYNVKLNELVNLVTDLQIGAFINPNGSIAPSKIEVGKLAFSTKTGESGAFINSEGQFRF DTLVYGDEKYGPLDIHIAAEHLDASALTVLKRKFAQISAKKMTEEQIRNDLIAAVKGEAS GLFTNNPVLDIKTFRFTLPSGKIDVGGKIMFKDMKKEDLNQLGLMLKKTEADIRMSIPQK MLEDLAVSQAGNIFSVNAEDEAEGRASLDDINETLRLMVDSTVQSMAREKYLTLNGDQID TAISLKNNQLKLNGKTLQNEPEPDFDEGGMVSEPQQ
Selection with vaccinees sera
Sera from the Meningococcal Reference Laboratory in Manchester has been made available to us. This sera has come from a clinical trial of OMV immunisation of volunteers.
Mutants selected by vaccinee Cl sera (screened once)
The following sequences were isolated
NMB0338 (as above) NMB0738 DNA sequence
ATGAAGATCGTCCTGATTAGCGGCCTGTCCGGTTCGGGCAAGTCCGTCGCACTGCGCCAA ATGGAAGATTCGGGTTATTTCTGCGTGGACAATTTGCCTTTGGAΆATGTTGCCCGCGCTG GTGTCGTATCATATCGAACGTGCGGACGAAACCGAATTGGCGGTCAGCGTCGATGTGCGT TCCGGCATTGACATCGGACAGGCGCGGGAACAGATTGCCTCTCTGCGCAGACTGGGGCAC AGGGTTGAAGTTTTGTTTGTCGAGGCGGAAGAAAGCGTGTTGGTCCGCCGGTTTTCCGAA
ACCAGGCGAGGACATCCTCTGAGCAATCAGGATATGACCTTGTTGGAAAGCTTAAAGAAA
GAΆCGGGAΆTGGCTGTTCCCGCTTAAAGAAATCGCCTATTGTΆTCGACACTTCCAAGATG AΆTGCCCAACAGCTCCGCCATGCAGTCCGGCAGTGGCTGAAGGTCGAACGTACCGGGCTG CTGGTGATTTTGGAGTCCTTCGGGTTCAAATACGGTGTGCCGΆACΆΆCGCGGATTTTATG TTCGΆTATGCGCAGCCTGCCCAΆCCCGTATTACGATCCCGAGTTGAGGCCTTACACCGGT ATGGACAΆGCCCGTTTGGGATTATTTGGACGGACAGCCGCTTGTGCAGGAAATGGTTGAC GACATCGAAAGGTTTGTTACGCATTGGTTACCGCGTTTGGAGGATGΆΆAGCAGGAGCTAC GTTACCGTCGCCATCGGTTGCACGGGAGGACAGCACCGTTCGGTCTATΆTTGTCGAAAAA CTCGCCCGAAGGTTGAAAGGGCGTTATGAATTGCTGATACGGCACAGACAGGCGCAAAAC CTGTCAGACCGCTAA
NMB0738 Protein sequence
MKIVLISGLSGSGKSVALRQMEDSGYFCVDNLPLEMLPALVSYHIERADETELAVSVDVR SGIDIGQAREQIASLRRLGHRVEVLFVEAEESVLVRRFSETRRGHPLSNQDMTLLESLKK EREWLFPLKEIAYCIDTSKMNAQQLRHAVRQWLKVERTGLLVILESFGFKYGVPNNADFM FDMRSLPNPYYDPELRPYTGMDKPVWDYLDGQPLVQEMVDDIERFVTHWLPRLEDESRSY VTVAIGCTGGQHRSVYIVEKLARRLKGRYELLIRHRQAQNLSDR NMB0792 NadC family (transporter) DNA sequence
ATGAACCTGCATGCAAAGGACAAAACCCAGCATCCCGAAAACGTCGAGCTGCTCAGTGCG CAGAAGCCGATTACCGACTTTAΆGGGCCTGCTGACCACCATTATTTCCGCCGTCGTCTGT TTCGGCATTTACCACATCCTGCCTTACAGCCCCGΆTGCCAATAΆAGGTATCGCGCTGCTG ATTTTCGTTGCCGCACTTTGGTTTACCGAGGCCGTCCACATTACCGTAΆCCGCACTGATG GTGCCGATTCTCGCCGTCGTACTCGGTTTCCCCGACATGGACATCAAAAAGGCGATGGCT GATTTTTCCAACCCGATTATCTACATTTTTTTCGGCGGCTTCGCGCTTGCCACCGCCCTG CATATGCAGCGGCTGGACCGTAAΆATCGCCGTCAGCCTGTTGCGCCTGTCGCGCGGCAΆT ATGAAΆGTGGCGGTTTTGATGTTGTTCCTCGTTACCGCCTTTCTGTCCATGTGGATCAGC AACACCGCCACCGCCGCGATGATGCTGCCTCTAGCAATGGGTATGCTGAGCCACCTCGAC CAGGAAAAAGAACACAAAACCTACGTCTTCCTCCTGCTCGGCATCGCCTATTGCGCCAGC ATCGGCGGCTTGGGCACGCTCGTCGGCTCGCCGCCCAACCTGATTGCCGCCAAAGCCCTA AATCTGGACTTCGTCGGCTGGATGAAGCTCGGCCTGCCGATGATGCTGTTGATTCTGCCC TTGATGCTGCTCTCCCTGTACGTCATCCTCAAACCTAATTTGAACGAACGCGTGGAΆΆTC AAΆGCCGAATCCATCCCTTGGACGCTGCACCGCGTGATCGCGCTGTTGATTTTCCTTGCC ACAGCCGCCGCGTGGATATTCAGCTCCAΆAΆTCAΆΆACCGCCTTCGGCATTTCCAATCCC GACACCGTTATCGCCCTGAGTGCCGCCGTCGCCGTCGTCGTCTTCGGCGTGGCGCAΆTGG AAGGAAGTCGCCCGCAATACCGACTGGGGCGTGTTGATGCTCTTCGGCGGCGGCATCAGC CTGAGCACGCTGTTGAAAACATCCGGCGCGTCCGAAGCCTTGGGACAGCAGGTTGCCGCC ACCTTTTCCGGCGCGCCCGCATTTTTGGTGATACTCΆTCGTCGCCGCCTTCATTATTTTT CTGACCGAGTTCACCAGCAACACCGCCTCCGCCGCATTGCTTGTACCGATTTTCTCCGGC ATCGCTATGCAGATGGGGCTGCCCGAACAAGTCTTGGTATTCGTCATCGGCATCGGCGCA TCTTGTGCCTTCATGCTGCCGGTTGCCACACCGCCTAACGCGATTGTGTTCGGCACGGGC TTAATCAAGCAΆCGCGAAATGATGAΆTGTCGGCATACTGCTGAΆCATCCTCTGCGTAGTA TTGGTTGCTCTGTGGGCTTATGCTGTACTGATGTAA
NMB0792 Protein sequence
MNLRAKDKTQHPENVELLSAQKPITDFKGLLTTIISAVVCFGIYHILPYSPDANKGIALL IFVAALWFTEAVHITVTALMVPILAWLGFPDMDIKKAMADFSNPIIYIFFGGFALATAL HMQRLDRKIAVSLLRLSRGNMKVAVLMLFLVTAFLSMWISNTATAAMMLPLAMGMLSHLD QEKEHKTYVFLLLGIAYCASIGGLGTLVGSPPNLIAAKALNLDFVGWMKLGLPMMLLILP LMLLSLYVILKPNLNERVEIKAESIPWTLHRVIALLIFLATAAΆWIFSSKIKTAFGISNP DTVIALSAAVAVWFGVAQWKEVARNTDWGVLMLFGGGISLSTLLKTSGASEALGQQVAA TFSGAPAFLVILIVAAFIIFLTEFTSNTASAALLVPIFSGIAMQMGLPEQVLVFVIGIGA SCAFMLPVATPPNAIVFGTGLIKQREMMNVGILLNILCWLVALWAYAVLM
NMB 0279 DNA sequence
ATGCAACGACAAATCAAACTGAAAAATTGGCTTCAGACCGTTTATCCCGAACGGGACTT1C GATCTGACTTTTGCGGCGGCGGATGCTGATTTCCGCCGCTATTTCCGTGCAACGTTTTCA GACGGCAGCAGTGTCGTCTGCATGGATGCACCGCCCGΆCΆAGATGAGTGTCGCACCTTAT TTGAAAGTGCAGAAACTGTTTGACATGGTCAATGTGCCGCAGGTATTGCACGCGGACACG GATCTGGGGTTTGTGGTATTGAACGACTTGGGCAATACGACGTTTTTGACCGCAATGCTT CAGGAΆCAGGGCGAAACGGCGCACAAΆGCCCTGCTTTTGGAGGCAATCGGCGAGTTGGTC GAATTGCAGAAGGCGAGCCGTGAAGGGGTTTTGCCCGAATATGACCGTGAAACGATGTTG CGCGAAATCAΆCCTGTTCCCGGAATGGTTTGTCGCAAΆAGΆATTGGGGCGCGΆATTAACA TTCAΆACAACGCCAACTTTGGCAGCAAACCGTCGATACGCTGCTGCCGCCCCTGTTGGCG CAGCCCAAAGTCTATGTGCACCGCGACTTTATCGTCCGCAACCTGATGCTGACGCGCGGC AGGCCGGGCGTTTTAGACTTCCAAGΆCGCGCTTTACGGCCCGATTTCCTACGATTTGGTG TCGCTGTTGCGCGATGCCTTTATCGAATGGGAAGAAGAATTTGTCTTGGACTTGGTTATC CGCTACTGGGAAAAGGCGCGGGCTGCCGGCTTGCCCGTCCCCGAAGCGTTTGACGAGTTT TACCGCTGGTTCGAATGGATGGGCGTGCAGCGGCACTTGAAGGTTGCAGGCATCTTCGCA CGCCTGTACTACCGCGACGGCAAAGΆCAAATACCGTCCGGAAATCCCGCGTTTCTTAAAC TATCTGCGCCGCGTATCGCGCCGTTATGCCGAACTCGCCCCGCTCTACGCGCTCTTGGTC GAACTGGTCGGCGATGAAGAACTGGAAACGGGCTTTACGTTTTAA
NMB0279 Protein sequence
MQRQIKLKNWLQTVYPERDFDLTFAAADADFRRYFRATFSDGSSVVCMDAPPDKMSVAPY LKVQKLFDMVNVPQVLHADTDLGFVVLNDLGNTTFLTAMLQEQGETAHKALLLEAIGELV ELQKASREGVLPEYDRETMLREINLFPEWFVAKELGRELTFKQRQLWQQTVDTLLPPLLA QPKVYVHRDFIVRNLMLTRGRPGVLDFQDALYGPISYDLVSLLRDAFIEWEEEFVLDLVI RYWEKARAAGLPVPEAFDEFYRWFEWMGVQRHLKVAGIFARLYYRDGKDKYRPEIPRFLN YLRRVSRRYAELAPLYALLVELVGDEELETGFTF NMB2050 DNA sequence
ATGGAACTGATGACTGTTTTGCTGCCTTTGGCGGCGTTGGTGTCGGGCGTGTTGTTTACA TGGTTGCTGATGAAGGGCCGGTTTCAGGGCGAGTTTGCCGGTTTGAACGCGCACCTGGCG GAΆAΆGGCGGCΆAGATGTGATTTTGTCGAACAGGCACACGGCAAAACCGTGTCGGAATTG GCGGTGTTGGACGGGAAATACCGGCATTTGCAGGACGAAAATTATGCTTTGGGCAACCGT TTTTCCGCAGCCGAAAAGCAGΆTTGCCCATTTGCAGGΆAAAΆGAGGCGGAGTCGGCGCGG CTGAΆGCAGTCGTATATCGAGTTGCAGGAAAΆGGCACAGGGTTTGGCGGTTGAAAACGAΆ CGTTTGGCAACGCAGCTCGGACAGGAACGGAAGGCGTTTGCCGACCAATATGCCTTGGAΆ CGCCAAATCCGCCAAΆGAATCGAAACCGATTTGGAAGAAΆGCCGCCAAΆCTGTCCGCGAC GTGCAAΆACGACCTTTCCGATGTCGGCAACCGTTTTGCCGCAGCCGAAAAACAGATTGCC CATTTGCAGGAAAAAGAGGCGGAAGCGGAGCGGTTGAGGCAGTCGCATACCGAGTTGCAG GAAAΆGGCACAGGGTTTGGCGGTTGAAAΆCGAACGTTTGGCAACGCAAΆTCGAACAGGAA CGCCTTGCTTCTGAAGAGAΆGCTGTCCTTGCTGGGCGAGGCGCGCAAAAGTTTGAGCGAT CAGTTTCAAAATCTTGCCAACACGATTTTGGAAGAAΆΆAAGCCGCCGTTTTACCGAGCAG AACCGCGAGCAGCTCCATCAGGTTTTGAACCCGCTAΆACGAACGCATCCACGGTTTCGGC GAGTTGGTCAAGCAAACCTATGATAAAGAATCGCGCGAGCGGCTGACGTTGGAAAACGAA TTGAAACGGCTTCAGGGGTTGAACGCGCAGCTGCACAGCGAGGCAAΆGGCCCTGACCAAC GCGCTGACCGGTACGCAGAATAAGGTTCAGGGCAATTGGGGCGAGATGATTCTGGAAACG GTTTTGGAAAATTCCGGCCTTCAGAAΆGGGCGGGAATATGTGGTTCAGGCGGCATCCGTC CGAAAAGAGGAAGACGGCGGCACGCGCCGCCTCCAGCCCGACGTTTTGGTCAACCTGCCC GACAACAAGCAGATTGTGATTGATTCCAAGGTCTCGCTGACAGCTTATGTGCGCTACACG CAGGCGGCGGATGCGGATACGGCGGCΆCGCGAACTGGCGGCACACGTTGCCAGCATCCGT GCACACATGAAAGGCTTGTCGCTGAAGGATTACACCGATTTGGAAGGTGTGAACACATTG GATTTCGTCTTTATGTTTATCCCTGTCGAACCGGCCTACCTGTTGGCGTTGCAGAATGAC GCGGGCTTGTTCCAAGAGTGTTTCGACAAΆCGGATTATGCTGGTCGGCCCCAGTACGCTG CTGGCGACTTTGAGGACGGTGGCGAATATTTGGCGCAACGAACAGCAAAATCAGAACGCA CTGGCGATTGCGGACGAAGGCGGCAAGCTGTACGACAAGTTTGTCGGCTTCGTACAGACG CTCGAAAGCGTCGGCAAAGGCATCGATCAGGCGCAAΆGCAGTTTTCAGACGGCATTCAAG • CAACTTGCCGAA.GGGCGCGGGAATCTGGTCGGACGCGCCGAGAAACTGCGTCTGTTGGGC GTGAAGGCAGGCAAACAACTTCAACGGGATTTGGTCGAGCGTTCCAATGAAACAACGGCG TTGTCGGAATCTTTGGAATACGCGGCAGAΆGATGAAGCAGTCTGA
NMB2050 Protein sequence MELMTVLLPLAALVSGVLFTWLLMKGRFQGEFAGLNAHLAEKAARCDFVEQAHGKTVSEL AVLDGKYRHLQDENYALGNRFSAAEKQIAHLQEKEAESARLKQSYIELQEKAQGLAVENE RLATQLGQERKAFADQYALERQIRQRIETDLEESRQTVRDVQNDLSDVGNRFAAAEKQIA HLQEKEAEAERLRQSHTELQEKAQGLAVENERLATQIEQERLASEEKLSLLGEARKSLSD QFQNLANTILEEKSRRFTEQNREQLHQVLNPLNERIHGFGELVKQTYDKESRERLTLENE LKRLQGLNAQLHSEAKALTNALTGTQNKVQGNWGEMILETVLENSGLQKGREYWQAASV RKEEDGGTRRLQPDVLVNLPDNKQIVIDSKVSLTAYVRYTQAADADTAΆRELAAHVASIR AHMKGLSLKDYTDLEGVNTLDFVFMFIPVEPAYLLALQNDAGLFQECFDKRIMLVGPSTL LATLRTVANIWRNEQQNQNALΆIADEGGKLYDKFVGFVQTLESVGKGIDQAQSSFQTAFK QLAEGRGNLVGRAEKLRLLGVKAGKQLQRDLVERSNETTALSESLEYAAEDEAV
NMB1335 CreA protein DNA sequence
ATGAACAGACTGCTACTGCTGTCTGCCGCCGTCCTGCTGACTGCCTGCGGCAGCGGCGAA ACCGATAAAATCGGACGGGCAAGTACCGTTTTCAΆCATACTGGGCAAAΆACGACCGTATC GAAGTGGAΆGGATTCGACGATCCCGACGTTCAAGGGGTTGCCTGTTATATTTCGTATGCA AAAAAAGGCGGCTTGAAGGAAATGGTCAATTTGGAAGAGGACGCGTCCGACGCATCGGTT TCGTGCGTTCAGACGGCATCTTCGATTTCTTTTGΆCGAAΆCCGCCGTGCGCAAACCGAAA GAΆGTTTTCAAACACGGTGCGAGCTTCGCGTTCAAGAGCCGGCAGATTGTCCGTTATTAC GACCCCAAACGCAAAACCTTCGCCTATTTGGTGTACAGCGATAAAATCATCCAAGGCTCG CCGΆΆAAATTCCTTAAGCGCGGTTTCCTGTTTCGGCGGCGGCATΆCCGCAAΆCCGATGGG GTGCAAGCCGATACTTCCGGCAACCTGCTTGCCGGCGCCTGCATGATTTCCAACCCGATA GAAAATCTCGACAAACGCTGA
NMB1335 Protein sequence
MNRLLLLSAAVLLTACGSGETDKIGRASTVFNILGKNDRIEVEGFDDPDVQGVACYISYA KKGGLKEMVNLEEDASDASVSCVQTASSISFDETAVRKPKEVFKHGASFAFKSRQIVRYY DPKRKTFAYLVYSDKIIQGSPKNSLSAVSCFGGGIPQTDGVQADTSGNLLAGACMISNPI ENLDKR
NMB2035 DNA sequence ATGACCGCCTTTGTCCACACCCTTTCΆGACGGCATGGAΆCTGACCGTCGAAΆTCAAGCGC CGTGCCAAGAAAAACCTGATTATCCGCCCCGCCGGCACACATACCGTCCGCATCAGCGTC CCACCCTGCTTCTCCGTCTCCGCTCTAAACCGCTGGCTGTATGAAAACGAAGCCGTCCTG CGGCAAACACTGGCGAAAACΆCCGCCGCCGCAAACTGCCGAAAACCGGCTGCCCGAATCC ATCCTCTTCCACGGCAGACAGCTTGCCCTCACCGCCCATCAAGACACGCAAATCCTGCTG ATGCCGTCTGAAATCCGTGTTCCCGAAGGCGCACCCGAAAAACAGCTTGCGCTGCTGCGG GACTTTTTGGAACGGCAGGCGCACAGTTACCTGATTCCCCGCCTCGAACGCCACGCCCGC ACCACACAACTGTTCCCCGCCTCCTCCTCGCTGACCTCTGCCAAAACCTTCTGGGGCGTG TGCCGCAAAΆCCACAGGCATACGCTTCAACTGGCGGCTGGTCGGCGCACCGGAΆTACGTT GCCGACTATGTCTGCATACACGAACTCTGCCACCTCGCCCATCCCGACCACAGCCCCGCC TTTTGGGAACTGACCCGCCGCTTCGCCCCCTACACGCCCAAAGCGAAACAGTGGCTCAAA ATCCACGGCAGGGAACTTTTCGCCTTAGGCTGA
NMB2035 Protein sequence
MTAFVHTLSDGMELTVEIKRRAKKNLIIRPAGTHTVRISVPPCFSVSALNRWLYENEAVL RQTLAKTPPPQTAENRLPESILFHGRQLALTAHQDTQILLMPSEIRVPEGAPEKQLALLR DFLERQAHSYLIPRLERHARTTQLFPASSSLTSAKTFWGVCRKTTGIRFNWRLVGAPEYV ADYVCIHELCHLAHPDHSPAFWELTRRFAPYTPKAKQWLKIHGRELFALG
NMB1351 Fmu and Fmvprotein DNA sequence ATGAACGCCGCACAACTCGACCATACCGCCAAAGTTTTGGCTGAAATGCTGACTTTCAAA CAGCCTGCCGATGCCGTCCTCTCCGCCTATTTCCGCGAACACAAAAAGCTCGGCAGTCAA GATCGCCACGAAATCGCCGAΆACCGCCTTTGCCGCGCTGCGCCACTATCAAAAAΆTCAGT ACCGCCCTACGCCGTCCGCACGCGCAGCCGCGCAAAGCCGCTCTCGCCGCACTGGTTCTC GGCAGAAGCACCAACATCAGCCAAATCAAAGACCTGCTTGATGAAGAAGAAACAGCGTTC CTCGGCAATTTGAAAGCCCGTAAAACCGAGTTTTCAGACAGCCTGAATACCGCCGCAGAA TTGCCGCAATGGCTGGTGGAACAACTGAAΆCAGCATTGGCGCGAAGAAGAAATCCTCGCT TTCGGCCGCAGCATCAACCAGCCTGCCCCGCTCGACATCCGCGTCAACACTTTGAAAGGC AAACGCGATAAAGTGCTGCCGCTGTTGCAAGCCGAAAGTGCCGATGCAGAGGCAACGCCT TATTCGCCTTGGGGCATCCGCCTGAAAAACAAAΆTCGCGCTTAACAAACACGAACTGTTT TTAGACGGCACACTGGAΆGTCCAAGACGAAGGCAGCCAGCTGCTTGCCTTATTGGTGGGC GCAAAACGAGGCGAAATCATTGTCGATTTCTGTGCCGGTGCCGGCGGTAAAACCTTGGCT GTCGGTGCGCAAATGGCGAACAAAGGCAGAATCTACGCCTTCGATATCGCCGAAAAACGC
CTTGCCAACCTCAAACCGCGTATGACCCGCGCCGGACTGACCAATATCCACCCCGAACGC
ATCGGCAGCGAACACGATGCCCGTATCGCCCGACTGGCAGGCAΆAGCCGACCGTGTGTTG GTGGACGCGCCCTGCTCCGGTTTGGGCACTTTACGCCGCAATCCCGACCTCAAATACCGC CAATCCGCCGAAΆCCGTCGCCAACCTTTTGGAACAGCAACACAGCATCCTCGATGCCGCC TCCAAACTGGTAAAACCGCAAGGACGTTTGGTGTACGCCACTTGCAGCATCCTGCCCGAA GAAAACGAGCTGCAAGTCGAACGTTTCCTGTCCGAACATCCCGAATTTGAACCCGTCAAC TGCGCCGAACTGCTTGCCGGTTTGAAAATCGATTTGGATACCGGCAAΆTACCTGCGCCTC AACTCCGCCCGACACCAAACCGΆCGGCTTCTTCGCCGCCGTATTGCAACGCAAΆTAA NMB1351 Protein sequence
MNAAQLDHTAKVLAEMLTFKQPADAVLSAYFREHKKLGSQDRHEIAETAFAALRHYQKIS
TALRRPHAQPRKAALAALVLGRSTNISQIKDLLDEEETAFLGNLKΆRKTEFSDSLNTAΆE
LPQWLVEQLKQHWREEEILAFGRSINQPAPLDIRVNTLKGKRDKVLPLLQAESADAEATP YSPWGIRLKNKIALNKHELFLDGTLEVQDEGSQLLALLVGAKRGEIIVDFCAGAGGKTLA VGAQMANKGRIYAFDIAEKRLANLKPRMTRAGLTNIHPERIGSEHDARIARLAGKADRVL VDAPCSGLGTLRRNPDLKYRQSAETVANLLEQQHSILDAASKLVKPQGRLVYATCSILPE ENELQVERFLSEHPEFEPVNCAELLAGLKIDLDTGKYLRLNSARHQTDGFFAAVLQRK
NMB1574 HvC DNA sequence ATGCAAGTCTATTACGATAAAGATGCCGATCTGTCCCTAATCAAAGGCAAAACCGTTGCC ATCATCGGTTACGGTTCGCAAGGTCATGCCCATGCCGCCAACCTGAAAGATTCGGGTGTA AACGTGGTGATTGGTCTGCGCCAAGGTTCTTCTTGGAAAΆAAGCCGAAGCAGCCGGTCAT GTCGTCAAAACCGTTGCTGAAGCGACCAAAGAAGCCGATGTCGTTATGCTGCTGCTGCCT GACGAAACCATGCCTGCCGTCTATCACGCCGAAGTTACAGCCAATTTGAAAGAAGGCGCA ACGCTGGCATTTGCACACGGCTTCAACGTGCACTACAACCAAATCGTTCCGCGTGCCGAC TTGGACGTGATTATGGTTGCCCCCAAAGGTCCGGGCCATACCGTACGCAGTGAATACAAA CGCGGCGGCGGCGTGCCTTCTCTGATTGCCGTTTACCAΆGACAATTCCGGCAAΆGCCAAA GACATCGCCCTGTCTTATGCGGCTGCCAACGGCGGCACCAAAGGCGGTGTGATTGAAACC ACTTTCCGCGAAGAAACCGAAACCGATCTGTTCGGCGAACAAGCCGTATTGTGCGGCGGC GTGGTCGAGTTGATCAAGGCGGGTTTTGAAACCCTGACCGAAGCCGGTTACGCGCCTGAA ATGGCTTACTTCGAATGTCTGCΆCGAAATGAΆACTGATCGTTGACCTGΆTTTTCGΆAGGC GGTATTGCCAATATGAACTACTCCATTTCCAACΆATGCGGAGTACGGCGAΆTACGTTACC GGCCCTGAAGTGGTCAATGCTTCCAGCAAAGΆAGCCATGCGCAATGCCCTGAAACGCATT CAAACCGGCGAATACGCAAAAATGTTTATCCAAGAGGGTAATGTCAACTATGCGTCTATG ACTGCCCGCCGCCGTCTGAATGCCGACCACCAAGTTGAAAAAGTCGGCGCACAACTGCGT GCCATGATGCCTTGGATTACTGCCAACAAATTGGTTGACCAAGACAAAAΆCTGA
NMB1574 Protein sequence
MQVYYDKDADLSLIKGKTVAIIGYGSQGHAHAANLKDSGVNVVIGLRQGSSWKKAEAAGH VVKTVAEATKEADVVMLLLPDETMPAVYHAEVTANLKΞGATLAFAHGFNVHYNQIVPRAD LDVIMVAPKGPGHTVRSEYKRGGGVPSLIAVYQDNSGKAKDIALSYAAANGGTKGGVIET TFREETETDLFGEQAVLCGGVVELIKAGFETLTEAGYAPEMΆYFECLHEMKLIVDLIFEG GIANMNYSISNNAEYGEYVTGPEVVNASSKEAMRNALKRIQTGEYAKMFIQEGNVNYASM TARRRLNADHQVEKVGAQLRAMMPWITANKLVDQDKN
NMB1298 rsuA DNA sequence
ATGAAACTTATCAAATACCTGCAATATCAAGGCATAGGAAGCCGCAAGCAGTGCCAATGG CTGATTGCCGGCGGTTATGTTTTCATCAACGGAΆCCTGCATGGACGACACCGATGCAGAC ATCGATTCCTCATCCGTCGAAACGTTGGATATTGACGGGGAAGCAGTAACCGTCGTTCCC
GAACCCTATTTCTΆCATCATGCTCΆΆCAAGCCTGAΆGATTΆCGAAΆCTTCGCACAAACCC
AΆGCACTACCGCΆGCGTATTCAGCCTGTTCCCCGACAΆTATGCGGAΆCATCGATATGCAG
GCGGTCGGCAGGCTGGATGCAGATACGACCGGCGTATTGCTGATTACCAACGACGGCAAA CTGAACCACAGCCTGACTTCGCCGAGCAGAΆAAΆTTCCCAAGCTGTΆCGAΆGTAACGCTC
AAACACCCCACAGGAGAAACGCTCTGCGAAACCTTGAAAAACGGCGTGCTGCTCCACGAC
GAAAΆCGΆAACCGTTTGTGCCGCCGATGCCGTTTTGAAAAACCCGACCACCCTGCTGCTG ACCATTACCGΆΆGGAAAATACCACCAΆGTCAΆACGCATGATCGCCGCCGCCGGCAACCGC GTGCAACACCTTCATCGCCGGCGATTCGCACATCTGGAAACAGAAAACCTCAAACCCGGG GΆATGGΆAATTTATCGAATGTCCAAΆATTCTGA
NMB1298 Protein sequence
MKLIKYLQYQGIGSRKQCQWLIAGGYVFINGTCMDDTDADIDSSSVETLDIDGEAVTWP EPYFYIMLNKPEDYETSHKPKHYRSVFSLFPDNMRNIDMQAVGRLDADTTGVLLITNDGK LNHSLTSPSRKIPKLYEVTLKHPTGETLCETLKNGVLLHDENETVCAADAVLKNPTTLLL TITEGKYHQVKRMIAAAGNRVQHLHRRRFAHLETENLKPGEWKFIECPKF
NMBl856 Lys R family (transcription regulator) DNA sequence
ATGΆAΆΆCCΆΆTTCAGAAGΆΆCTGACCGTATTTGTTCAAGTGGTGGAAΆGCGGCAGCTTC AGCCGTGCGGCGGAGCAGTTGGCGATGGCAAATTCTGCCGTAAGCCGCATCGTCAAACGG
CTGGAGGAAAAGTTGGGTGTGAΆCCTGCTCAΆCCGCACCACGCGGCAACTCAGTCTGACG
GΆΆGAΆGGCGCGCAATATTTCCGCCGCGCGCAGAGAATCCTGCAAGAAATGGCAGCGGCG
GAAΆCCGAAATGCTGGCAGTGCACGAAΆTACCGCAAGGCGTGTTGAGCGTGGATTCCGCG
ATGCCGΆTGGTGCTGCATCTGCTGGCGCCGCTGGCAGCAAAATTCAΆCGAACGCTATCCG CATATCCGACTTTCGCTCGTTTCTTCCGAAGGCTATATCAATCTGATTGAACGCAAAGTC
GATATTGCCTTACGGGCCGGAGAATTGGACGATTCCGGGCTGCGTGCACGCCATCTGTTT
GACAGCCGCTTCCGCGTAATCGCCAGTCCTGAΆTACCTGGCAAAACACGGCACGCCGCAΆ
TCTACAGAAGAGCTTGCCGGCCACCAATGTTTAGGCTTCACCGAACCCGGTTCTCTAAAT
ACATGGGCGGTTTTAGATGCGCAGGGAAATCCCTATAΆGATTTCACCGCACTTTACCGCC AGCAGCGGTGAAATCTTACGCTCGTTGTGCCTTTCAGGTTGCGGTATTGTTTGCTTATCA GATTTTTTGGTTGACAACGACATCGCTGAAGGAAAGTTAATTCCCCTGCTCGCCGAACAA ACCTCCGΆTAΆAACACACCCCTTTΆATGCTGTTTATTACAGCGATAAAGCCGTCAATCTC CGCTTACGCGTATTTTTGGATTTTTTAGTGGAGGΆΆCTGGGΆAΆCAATCTCTGTGGATAA NMB1856 Protein sequence
MKTNSEELTVFVQWESGSFSRAAEQLAMANSAVSRIVKRLEEKLGVNLLNRTTRQLSLT EEGAQYFRRAQRILQEMAAAETEMLAVHEIPQGVLSVDSΆMPMVLHLLAPLAAKFNERYP HIRLSLVSSEGYINLIERKVDIALRΆGELDDSGLRARHLFDSRFRVIASPEYLAKHGTPQ STEELAGHQCLGFTEPGSLNTWAVLDAQGNPYKISPHFTASSGEILRSLCLSGCGIVCLS DFLVDNDIAEGKLIPLLAEQTSDKTHPFNAVYYSDKAVNLRLRVFLDFLVEELGNNLCG
NMBOl 19 DNA sequence
ATGATGAAGGATTTGAATTTGAGCAACAGCCTGTTCAAAGGCTACAACGACAAACATGGC TTAATGATTTGTGGCTATGAATGGGGTTGGAGTAAAGCCGATGAGGCTGCTTATGTAGCA GGTGAATACAAACTCCCTGAAAACAAAATCGACCATACATTTGCAAACAAATCCCTCTAT TTCGGAGAGCAGGCAAAAΆAGTGGCGTTACGACAATACGATAAAAAΆTTGGTTTGΆAATG TGGGGACACCCCTTAGACGAAAATGGATTGGGCGGTGCATTTGAAAΆΆTCCCTGGTTCAA ACCAACTGGGCTGCTACACAGGGCAACACTATCGACAATCCCGACAAGTTCACACAACCC GAGCACATCGATAATTTTCTCTACCACATCGAAΆAACTGCGTCCGAAΆGTCATCCTCTTC ATGGGCAGCAGGTTGGCGGATTTTCTGAACAACCAAAATGTACTGCCACGCTTCGAGCAG TTGGTCGGTAAGCAGACCAAACCGCTGGAGACGGTGCAAAAAGAATTTGACGGTACACGT TTCAΆTGTCAAATTCCAATCGTTTGAAGΆTTGCGAΆGTCGTCTGCTTTCCCCATCCCAGT GCCAGTCGCGGTCTATCTTACGATTACATCGCCTTGTTTGCGCCTGAAATGAACCGGATT TTATCGGACTTTAAAACAACACGCGGATTCAAATAA
NMBOl 19 Protein sequence
MMKDLNLSNSLFKGYNDKHGLMICGYEWGWSKADEAAYVAGEYKLPENKIDHTFANKSLY FGEQAKKWRYDNTIKNWFEMWGHPLDENGLGGAFΞKSLVQTNWAATOGNTIDNPDKFTQP EHIDNFLYHIEKLRPKVILFMGSRLADFLNNQNVLPRFEQLVGKQTKPLETVQKEFDGTR FNVKFQSFEDCEVVCFPHPSASRGLSYDYIALFAPEMNRILSDFKTTRGFK NMB 1705 rfaK DNA sequence
ATGGAAAAAGAATTCAGGATATTAAATATCGTATCGGCCAAGATTTGGGGTGGAGGCGAA CAATATGTCTATGATGTTTCAAAAGCATTGGGGCTTCGGGGCTGCACAATGTTTACCGCC
GTCAATAAAAΆTAATGAΆTTGATGCACAGGCGATTTTCCGAAGTTTCTTCCGTTTTCACA
ACGCGCCTTCACACGCTCAACGGGCTGTTTTCGCTCTACGCACTTACCCGCTTTATCCGG AAAAACCGCATTTCCCACCTGATGATACACACCGGCAAAATTGCCGCCTTATCCATACTT TTGAAAAAACTGACCGGGGTGCGCCTGATΆTTTGTCAAACATAATGTCGTCGCCAACAAA ACCGATTTTTACCACCGCCTGATACAGAAAAACACAGACCGCTTTATTTGCGTTTCCCGT CTGGTTTACGATGTGCAAACCGCCGACAATCCCTTTAAAGAAAAATΆCCGGATTGTTCAT AACGGTATCGATACCGGCCGTTTCCCTCCCTCTCAΆGAAAAACCCGACAGCCGTTTTTTT ACCGTCGCCTACGCCGGCAGGATCAGTCCAGAAAAAGGATTGGAAAACCTGATTGAAGCC TGTGTGATACTGCATCGGAAΆTΆTCCTCAΆATCAGGCTCAAATTGGCAGGGGACGGACAT CCGGATTATATGTGCCGCCTGAAGCGGGACGTATCTGCTTCAGGAGCAGAACCATTTGTT TCTTTTGAAGGGTTTACCGAAAAΆCTTGCTTCGTTTTACCGCCAAΆGCGATGTCGTGGTT TTGCCCAGCCTCGTCCCGGAGGCATTCGGTTTGTCATTATGCGAGGCGATGTACTGCCGA ACGGCGGTGATTTCCAATACTTTGGGGGCGCAAAAGGAAATTGTCGAACATCATCAATCG GGGATTCTGCTGGACAGGCTGACACCTGAΆTCTTTGGCGGACGAAATCGAΆCGCCTCGTC TTGAACCCTGAAΆCGAAAΆΆCGCACTGGCΆACGGCAGCTCATCAATGCGTCGCCGCCCGT TTTACCATCAACCATACCGCCGACAAΆTTATTGGATGCAATATAA NMB1705 Protein sequence
MEKEFRILNIVSAKIWGGGEQYVYDVSKALGLRGCTMFTAVNKNNELMHRRFSEVSSVFT TRLHTLNGLFSLYALTRFIRKNRISHLMIHTGKIAALSILLKKLTGVRLIFVKHNWANK TDFYHRLIQKNTDRFICVSRLVYDVQTADNPFKEKYRIVHNGIDTGRFPPSQEKPDSRFF TVAYAGRISPEKGLENLIEACVILHRKYPQIRLKLAGDGHPDYMCRLKRDVSASGAEPFV SFEGFTEKLASFYRQSDVWLPSLVPEAFGLSLCEAMYCRTAVISNTLGAQKEIVEHHQS GILLDRLTPΞSLADEIERLVLNPETKNALATAAHQCVAARFTINHTADKLLDAI
NMB2065 Hemicprotein DNA sequence
ATGCAGGAACAGAATCGGAAACCAAGTTTTCCCATAGTGATGTTGCTGGTGTCGGTTGCC CTGTGGATAGCGTCTTTATCCAATGTTGCATTTTATTTGGGCAATCATGGAAGCATGGAG GGTTTGACCGTTTTGATTTTGGGGTCGATATTTGCTTCTTTGGATATCAGGTATTGTGCG GTCTATGCGAATTATGTTTGGTTGGCGGCCATTGTTTTGCTGGCGTTGCGGAAGAAGGTC GTGCCTGTCCATGCGGCACTTTGGGGCTTGGCGTTGGTGGCTTTCAGTGTGAAAGCCGTA
TACGTCGATGAAGCAGGGAATACΆTCGGATATTGTGCGCTACGGTGCAGGATTTTATTTG TGGTATGCCGCATTTGCGGTTGCCACCATCGGTACGTTTGCCGGAAAGAATAAGGAAAGA AAAGCCGCATCAGCGGCAGACGGGATAAAAATGACGTTTGATAAATGGTTGGGCTTGTCA AAACTGCCTAAAAATGAΆGCAAGAΆTGCTGCTACAATATGTTTCGGAATATACGCGCGTG CAGTTGTTGACGCGGGGCGGGGAAGAAATGCCGGACGAAGTCCGACAGCGGGCGGACAGG CTGGCGCAACGCCGTCTGAΆCGGCGAGCCGGTTGCCTATATTTTAGGTGTGCGCGAΆTTT TATGGCAGACGCTTTACAGTCAATCCGAGCGTGCTGATTCCGCGCCCCGAAACCGAACAT TTGGTCGAAGCCGTATTGGCGCGCCTGCCCGAAAACGGGCGCGTGTGGGATTTGGGGACG GGCAGCGGCGCGGTTGCCGTAACCGTCGCGCTCGAACGCCCCGATGCGTTTGTGCGCGCA TCCGACATCAGCCCGCCCGCCCTTGAAACGGCGCGGAAAAATGCGGCGGATTTGGGCGCG CGGGTCGAATTTGCACACGGTTCGTGGTTCGACACCGATATGCCGTCTGAAGGGAAATGG GACATCATCGTGTCCAACCCGCCCTATATCGAAAACGGCGATAAACATTTGTTGCAAGGC GATTTGCGGTTTGAGCCGCAAATCGCGCTGACCGACTTTTCAGACGGCCTAAGCTGCATC CGCACCTTGGCGCAAGGCGCGCCCGACCGTTTGGCGGAAGGCGGTTTTTTATTGCTGGAA CACGGTTTCGATCAGGGCGCGGCGGTGCGCGGCGTGTTGGCGGAGAΆTGGTTTTTCAGGA GTGGAAACCCTGCCGGATTTGGCGGGTTTGGACAGGGTTACGCTGGGGAAGTATATGAAG CATTTGAAATAA NMB2065 Proteinsequence
MQEQNRKPSFPIVMLLVSVALWIASLSNVAFYLGNHGSMEGLTVLILGSIFASLDIRYCΆ VYANYVWLAAIVLLALRKKVVPVHAALWGLALVAFSVKAVYVDEAGNTSDIVRYGAGFYL WYAAFAVATIGTFAGKNKERKAASAADGIKMTFDKWLGLSKLPKNEARMLLQYVSEYTRV QLLTRGGEEMPDEVRQRADRLAQRRLNGEPVAYILGVREFYGRRFTVNPSVLIPRPETEH LVEAVLARLPENGRVWDLGTGSGAVAVTVΆLERPDAFVRASDISPPALETΆRKNAADLGA RVEFAHGSWFDTDMPSEGKWDIIVSNPPYIENGDKHLLQGDLRFEPQIALTDFSDGLSCI RTLAQGAPDRLAEGGFLLLEHGFDQGAAVRGVLAENGFSGVETLPDLAGLDRVTLGKYMK
HLK
Mutants selected by vacinee's 17 D sera (Screened once only)
NMB0339 DNA sequence
ATGGACAACGAATTGTGGATTATCCTGCTGCCGATTATCCTTTTGCCCGTCTTCTTCGCG ATGGGCTGGTTTGCCGCCCGCGTGGATATGAAAACCGTATTGAAGCAGGCAAΆAΆGCATC CCTTCGGGATTTTATAAAΆGCTTGGACGCTTTGGTCGACCGCAACAGCGGGCGCGCGGCA AGGGAGTTGGCGGAAGTCGTCGΆCGGCCGGCCGCΆATCGTATGATTTGAΆCCTCΆCCCTC GGCAAACTTTACCGCCΆGCGTGGCGAAAΆCGACA-AAGCCATCAΆCATACACCGGACAATG CTCGATTCTCCCGATACGGTCGGCGAAAAGCGCGCGCGCGTCCTGTTTGAATTGGCGCΆΆ AACTACCAAAGTGCGGGGTTGGTCGATCGTGCCGAACAGATTTTTTTGGGGCTGCAAGAC GGTAAAATGGCGCGTGAΆGCCAGACAGCACCTGCTCAATATCTACCAACAGGACAGGGAT TGGGAAAAAGCGGTTGAAACCGCCCGGCTGCTCAGCCATGACGATCAGACCTATCAGTTT GΆAΆTCGCCCAGTTTTATTGCGAACTTGCCCAAGCCGCGCTGTTCΆAGTCCAATTTCGAT GTCGCGCGTTTCAATGTCGGCAAGGCACTCGAAGCCAACAAAAAATGCACCCGCGCCAAC ATGATTTTGGGCGACATCGAACACCGACAAGGCAATTTCCCTGCCGCCGTCGAAGCCTAT GCCGCCATCGAGCAGCAAΆACCATGCΆTACTTGAGCATGGTCGGCGAGAAGCTTTACGAA GCCTATGCCGCGCAGGGAAΆACCTGAAGAAGGCTTGAACCGTCTGΆCΆGGATATATGCAG ACGTTTCCCGAACTTGACCTGATCAΆTGTCGTGTACGAGΆAATCCCTGCTGCTTAΆGTGC GAGAAAGAAGCCGCGCAΆACCGCCGTCGAGCTTGTCCGCCGCAAGCCCGACCTTAACGGC GTGTACCGCCTGCTCGGTTTGAAACTCAGCGATATGAATCCGGCTTGGAAAGCCGATGCC GACATGATGCGTTCGGTTATCGGACGGCAGCTACAGCGCAGCGTGATGTACCGTTGCCGC
AACTGCCACTTCAAATCCCAAGTCTTTTTCTGGCACTGCCCCGCCTGCAACAAATGGCAG
ACGTTTACCCCGAATAAΆATCGAΆGTTTAA NMB 0339 Protein sequence
MDNELWIILLPIILLPVFFAMGWFAARVDMKTVLKQAKSIPSGFYKSLDALVDRNSGRAA RELAEVVDGRPQSYDLNLTLGKLYRQRGENDKAINIHRTMLDSPDTVGEKRARVLFELAQ NYQSAGLVDRΆEQIFLGLQDGKMΆREARQHLLNIYQQDRDWEKAVETARLLSHDDQTYQF EIAQFYCELΆQAALFKSNFDVARFNVGKALEANKKCTRANMILGDIEHRQGNFPAAVEAY AAIEQQNHAYLSMVGEKLYEAYAAQGKPEEGLNRLTGYMQTFPELDLINWYEKSLLLKC EKEAAQTAVELVRRKPDLNGVYRLLGLKLSDMNPAWKADADMMRSVIGRQLQRSVMYRCR NCHFKSQVFFWHCPACNKWQTFTPNKIEV
Selection -with patient's sera We have a collection of acute and convalescent sera available to us for screening. This is from individuals infected with different serogroup of TV. meningitidis. Screens have been performed with acute (A) or convalescent (C) sera. The period between the acute infection and collection of sera was from 2 weeks to 3 months.
NMB0401 putA DNA sequence
ATGTTTCATTTTGCATTTCCGGCACAAACTGCCCTGCGCCAAGCGATAACCGATGCCTAC CGCCGTAATGAAΆTCGΆAGCCGTACAGGATATGTTGCAACGTGCACAGATGAGCGACGAA GAGCGCAACGCCGCCTCCGAGCTTGCCCGCCGTTTGGTTACCCAAGTCCGCGCCGGCCGC ACCAAAGCCGGCGGCGTGGATGCGCTGATGCACGAGTTTTCACTCTCCAGCGAAGAAGGC ATCGCGCTGATGTGTCTGGCAGAAGCCCTGCTGCGTATCCCCGACAACGCCACGCGCGAC CGCCTGATTGCCGACAΆGATTTCAGACGGCAACTGGAAAAGCCATTTGAACAACAGCCCT TCCCTCTTCGTCAATGCTGCCGCCTGGGGCCTGCTGATTACCGGCAAACTGACCGCCACA
AACGACAAACΆAATGAGTTCCGCACTCAGCCGCCTGATCAGCAAAGGCGGCGCACCGCTC ATCCGCCAAGGCGTAAATTACGCCATGCGGCTTCTGGGCAAACAGTTCGTAACCGGACAG ACCATTGAAGAAGCCCTGCAAAACGGCAAAGAACGCGAAAAAΆTGGGCTACCGCTTCTCC TTCGATATGTTGGGCGAAGCCGCCTACACCCAAGCCGATGCCGACCGCTACTACCGCGAC TATGTCGAAGCCATCCACGCCATCGGCAAAGATGCGGCAGGACAAGGCGTTTACGAAGGT AACGGTATTTCCGTCAAACTTTCCGCCATCCATCCGCGCTACTCGCGCΆCCCAACACGGC CGCGTGATGGGCGAACTGTTGCCGCGCCTGAAAGAGCTGTTCCTTTTGGGTAAΆΆΆATAC GATATCGGTATCAACATCGATGCCGAAGΆAGCCAACCGTCTGGAGCTGTCTTTGGATTTG ATGGAGGCTTTGGTTTCAGACCCTGACTTGGCTGGCTACAAAGGTATCGGTTTCGTTGTC CAAGCCTACCAAAAACGTTGTCCGTTCGTTATCGACTACCTGATCGACCTTGCCCGCCGC AACAACCAAΆAACTAATGATCCGCCTCGTCAAAGGCGCGTATTGGGACAGCGAAATCAAA TGGGCGCAAGTGGACGGCTTGAACGGCTATCCGACCTACACCCGCAAAGTCCACACCGAC ATCTCCTACCTCGCCTGCGCGCGCAΆACTGCTTTCCGCGCAAGACGCGGTATTCCCGCAA TTTGCCACCCACAACGCCTACACTTTGGGCGCΆΆTCTACCAAATGGGTAAAGGCAΆAGAT TTTGAACACCAATGCCTGCACGGTATGGGCGAAACCCTGTACGACCAAGTCGTCGGCCCG CAAAACTTAGGCCGCCGCGTGCGCGTGTACGCCCCAGTCGGCACACACGAAACCCTGCTC GCCTACTTGGTGCGCCGCCTGTTGGAAAACGGCGCGAACTCGTCTTTCGTCAΆCCAAATC GTCGATGAAΆACATCAGCATCGACACGCTCATCCGCAGCCCGTTCGACACCATCGCCGAA CAAGGCATCCACCTGCACAACGCCCTGCCGCTGCCGCGCGATTTGTΆCGGCAAATGCCGT CTGAACTCGCAAGGCGTGGACTTGAGCAACGAAAACGTATTGCAGCAGCTTCAAGAACAG ATGAACAAAGCCGCCGCGCAAGACTTCCACGCCGCATCCATCGTCAACGGCAAAGCCCGC GATGTCGGCGAAGCGCAACCGATTAAAAACCCTGCCGACCACGACGACATCGTCGGCACA GTCAGCTTTGCCGATGCCGCGCTTGCCCAAGAAGCGGTTGGCGCAGCCGTTGCCGCGTTC CCCGAATGGAGTGCGACACCTGCCGCCGAACGCGCCGCCTGCCTGCGCCGTTTTGCCGΆT TTGCTGGAGCAGCACACCCCAGCACTGATGATGCTTGCCGTGCGCGAAGCAGGCAAAACG CTGAΆCAACGCCATTGCCGAAGTGCGCGAAGCCGTCGATTTCTGCCGCTACTACGCΆAAC GAAGCCGAACATACCCTGCCTCAAGACGCAAAAGCCGTCGGCGCGATTGTCGCCATCAGC CCGTGGAACTTCCCGCTCGCCATCTTTACCGGCGAAGTCGTTTCCGCATTGGCGGCAGGC ΆACACCGTCATCGCCAAACCCGCCGAACAAACCAGCCTGATTGCCGGTTATGCCGTTTCC CTCATGCACGAAGCCGGCATCCCGACTTCCGCCCTGCAACTCGTCCTCGGCGCAGGCGAC GTGGGTGCGGCATTGACCAACGATGCCCGCATCGGCGGCGTGATTTTCACCGGCTCGACC GAAGTGGCGCGCCTGATCAACAAAGCCCTTGCCAAACGCGGCGACAATCCCGTCCTGATT GCCGAAACCGGCGGACAAAΆCGCCATGATTGTCGATTCCACCGCACTTGCCGAGCAAGTC TGCGCCGACGTATTGAACTCCGCCTTCGACAGCGCGGGACAACGCTGCTCCGCCCTGCGC ATTTTGTGCGTCCAAGAAGACGTTGCCGACCGTATGCTCGACATGATCAAAGGCGCTATG GACGAΆCTCGTCGTCGGCAAACCGATTCAGCTCACTACCGATGTCGGCCCCGTCATCGAT GCCGAAGCACAGCAAAACCTGTTGAACCACATCAACAAAATGAAAGGTGTTGCCAAGTCC TACCACGAΆGTCAAAΆCCGCCGCCGATGTCGATTCCAAAAAATCCACGTTCGTTCGCCCC ATCCTGTTTGAATTGAACAACCTCΆACGAACTGCAACGCGAAGTCTTCGGTCCCGTCCTG CACGTCGTCCGCTACCGCGCCGACGAACTCGACAACGTCATCGACCAAATCAACAGCAAA GGCTACGCCCTGACCCACGGCGTACACAGCCGCATCGAAGGCACGGTACGCCACATCCGC AGCCGCATCGAAGCCGGCAACGTTTACGTCAACCGCAACATCGTCGGCGCAGTCGTCGGC GTACAGCCCTTCGGCGGACACGGTCTGTCCGGCACAGGCCCCAAAGCAGGCGGTTCGTTC TACCTGCAAAAACTGACCCGCGCCGGCGAATGGGTTGCCCCGACCCTGAGCCAAΆTCGGA CAGGCGGACGAAGCCGCACTCAAACGCCTCGAAGCACTGGTTCACAAACTACCGTTCAAC GCCGAAGAGAAAAAAGCCGCAGCGGCCGCTTTGGGACACGCCCGCATCCGCACCCTGCGC CGTGCCGAAACCGTCCTTACCGGACCGACCGGCGAGCGCAACAGCATCTCATGGCACGCG CCCAAACGCGTTTGGATACACGGCGGCAGCACGGTTCAAGCCTTTGCCGCACTGACCGAA CTTGCCGCCTCCGGCATACAGGCAGTGGTCGAACCCGACAGCCCCTTGGCTTCCTACACT GCCGACTTGGAAGGTCTGCTGCTGGTCAACGGCAAACCCGAZVACCGCCGGCATCAGCCAC GTTGCCGCCCTGTCGCCTTTGGACAGCGCGCGCAAACAGGAACTTGCCGCCCACGACGGC GCACTCATCCGCATCCTCCCTTCGGAAAACGGACTCGACATCCTGCAAGTGTTTGAΆGAA ATCTCTTGCAGCGTCAACACCACAGCCGCCGGCGGCAACGCCAGCCTGATGGCGGTCGCC GAC T GA
NMB0401 Proteinsequence
MFHFAFPAQTALRQAITDAYRRNEIEAVQDMLQRAQMSDEERNAASELARRLVTQVRAGR TKAGGVDALMHEFSLSSEEGIALMCLAEALLRIPDNATRDRLIADKISDGNWKSHLNNSP SLFVNAAAWGLLITGKLTATNDKQMSSALSRLISKGGΆPLIRQGVNYAMRLLGKQFVTGQ TIEEALQNGKEREKMGYRFSFDMLGEAAYTQADADRYYRDYVEALHAIGKDAAGQGVYEG NGISVKLSAIHPRYSRTQHGRVMGELLPRLKELFLLGKKYDIGINIDAEEANRLELSLDL
MEALVSDPDLAGYKGIGFVVQΆYQKRCPFVIDYLIDLARRNNQKLMIRLVKGAYWDSEIK WAQVDGLNGYPTYTRKVHTDISYLACARKLLSAQDAVFPQFATHNAYTLGAIYQMGKGKD FEHQCLHGMGETLYDQVVGPQNLGRRVRVYAPVGTHETLLAYLVRRLLENGANSSFVNQI VDENISIDTLIRSPFDTIAEQGIHLHNALPLPRDLYGKCRLNSQGVDLSNENVLQQLQEQ MNKAAAQDFHAΆSIVNGKΆRDVGEAQPIKNPADHDDIVGTVSFADΆALAQEAVGΆAVAΆF PEWSATPAAERΆACLRRFADLLEQHTPΆLMMLAVREAGKTLNNAIAEVREAVDFCRYYAN EAEHTLPQDAKAVGAIVAISPWNFPLAIFTGEVVSALAAGNTVIAKPAEQTSLIAGYAVS LMHEAGIPTSALQLVLGAGDVGAALTNDARIGGVIFTGSTEVARLINKALAKRGDNPVLI AETGGQNAMIVDSTALAEQVCADVLNSAFDSAGQRCSALRILCVQEDVADRMLDMIKGAM DELWGKPIQLTTDVGPVIDAEAQQNLLNHINKMKGVAKSYHEVKTAADVDSKKSTFVRP ILFELNNLNELQREVFGPVLHVVRYRADELDNVIDQINSKGYALTHGVHSRIEGTVRHIR SRIEAGNVYVNRNIVGAWGVQPFGGHGLSGTGPKAGGSFYLQKLTRAGEWVAPTLSQIG QADEAALKRLEALVHKLPFNAEEKKAAΆAALGHARIRTLRRAETVLTGPTGERNSISWHA PKRVWIHGGSTVQAFAALTELAASGIQAWEPDSPLASYTADLEGLLLVNGKPETAGISH VAALSPLDSARKQELAAHDGALIRILPSENGLDILQVFEEISCSVNTTAAGGNASLMAVA
D
NMB1335 CreA
DNA andProtein sequences given above
NMB1467 PPXDNA sequence
ATGACCACCACCCCCGCAAACGTCCTCGCCTCCGTCGATTTGGGTTCCAACAGTTTCCGC CTCCAGATTTGCGΆAAACAACAACGGACAΆTTAAAΆGTCATCGATTCGTTCAΆACAGATG GTGCGCTTCGCCGCCGGACTGGACGAACAGAAAAΆTCTGAGTGCCGCTTCCCAAGAACAG GCTTTGGACTGTCTGGCAAAATTCGGCGAΆCGCCTGCGCGGCTTCCGCCCTGAACAGGTA CGCGCCGTGGCAACCAACACATTCCGCGTTGCCAAAAACATCGCAGATTTCCTTCCCAAA GCCGAAGCGGCATTGGGTTTCCCCATCGAAATCATCGCCGGGCGCGΆAGAGGCGCGGCTG ΆTTTATACCGGCGTGATCCACACCCTCCCCCCGGGCGGCGGCAAAATGCTGGTTΆTCGAC ATCGGCGGCGGTTCGACAGAATTTGTCATCGGCTCGACGCTGAATCCCGACATTACCGAA AGCCTGCCCTTGGGCTGCGTAACCTACAGCCTGCGCTTCTTCCAAAACΆAAATCACCGCC AAAGACTTCCAATCTGCCATTTCCGCCGCCCGCAACGAAATCCAGCGTATCAGCAAAAAT ATGAGGCGCGAAGGTTGGGATTTCGCCGTCGGCACATCGGGTTCGGCAAAATCCATCCGC GACGTGCTTGCCGCCGAAATGCCCCAAGAGGCGGACATTACCTACΆAΆGGCATGCGCGCC CTCGCCGAACGCΆTCATCGAAGCCGGTTCGGTCAAAAAAGCCAAATTTGAAAACCTGAAA CCGGAACGCΆTCGAAGTTTTTGCCGGCGGACTTGCCGTGATGATGGCGGCGTTTGAGGAA ATGAAACTCGACAGGATGACCGTAACCGAAGCCGCCCTGCGCGACGGCGTGTTTTACGAT TTGATCGGGCGCGGTTTAAACGAAGATATGCGCGGACAAΆCGGTTGCCGAGTTCCAACAC CGCTACCACGTCAGCCTCΆATCAGGCGAAΆCGCACCGCCGAGACCGCGCAAACCTTTATG GACΆGCCTCTGCCACGCTAAAAACGTTACAGTTCAAGAGCTTGCCTTGTGGCAACAGTAT CTCGGACGCGCCGCCGCGCTGCACGAAATCGGTTTGGΆCATCGCCCACACCGGCTATCAC AAGCATTCCGCCTACATCCTCGAAAACGCCGATATGCCGGGTTTCTCACGCAAAGAACAG ACCATACTTGCCCAACTGGTCATCGGTCATCGCGGCGATATGAAAAAAATGAGCGGCATC ATCGGCACCAACGAAATGTTGTGGTATGCCGTTTTGTCCCTGCGCCTTGCCGCACTGTTC TGCCGTTCGCGCCAAGACCTGTCTTTCCCGAAAAATATGCAGTTGCGCACGGATACGGAA AGCTGCGGCTTCATCCTGCGTATTGACAGGGAΆTGGCTGGAACGCCATCCCCTGATTGCC GACGCATTGGAATATGAAAGCGTCCAATGGCAAAAΆATCAΆTATGCCGTTCΆAAGTCGAG GCCGTCTGA
NMB1467 Protein sequence
MTTTPANVLASVDLGSNSFRLQICENNNGQLKVIDSFKQMVRFAAGLDEQKNLSAASQEQ ALDCLAKFGERLRGFRPEQVRAVATNTFRVAKNIADFLPKAEAALGFPIEIIAGREEARL IYTGVIHTLPPGGGKMLVIDIGGGSTEFVIGSTLNPDITESLPLGCVTYSLRFFQNKITA KDFQSAISAARNEIQRISKNMRREGWDFAVGTSGSAKSIRDVLAAEMPQEADITYKGMRA LAERIIEAGSVKKAKFENLKPERIEVFAGGLAVMMAAFEEMKLDRMTVTEAALRDGVFYD LIGRGLNEDMRGQTVAEFQHRYHVSLNQAKRTAETAQTFMDSLCHAKNVTVQELALWQQY LGRAAALHEIGLDIAHTGYHKHSAYILENADMPGFSRKEQTILAQLVIGHRGDMKKMSGI IGTNEMLWYAVLSLRLAALFCRSRQDLSFPKNMQLRTDTESCGFILRIDREWLERHPLIA DALEYESVQWQKINMPFKVEAV
NMB2056 HemIC
ATGAACGGTAAATACTACTACGGCACAGGCCGCCGCAAAAGTTCAGTGGCTCGTGTATTC CTGΆTTAAΆGGTACAGGTCAAΆTCATCGTAAΆCGGTCGTCCCGTTGACGΆATTCTTCGCA CGGGAAACCAGCCGAATGGTTGTTCGCCAACCCTTGGTTCTGACTGAAAΆCGCCGΆΆTCT TTCGACATCAAAGTCAATGTTGTTGGCGGCGGCGAAACCGGCCAGTCCGGCGCAATCCGC CACGGCATTACCCGTGCCCTGATCGACTTCGATGCCGCGTTGAAACCCGCCTTGTCTCAA GCTGGTTTTGTTACCCGCGΆTGCCCGCGAAGTCGAACGTAAAΆAACCGGGTCTGCGCAAA GCACGCCGTGCAAAACAATTCTCCAAACGTTAA
NMB2056 Protein sequence MNGKYYYGTGRRKSSVARVFLIKGTGQIIVNGRPVDEFFARETSRMWRQPLVLTENAES FDIKVNVVGGGETGQSGAIRHGITRALIDFDAALKPALSQAGFVTRDARΞVERKKPGLRK
ARRAKQFSKR
NMB0808 DNA sequence ATGTCCGCCCTCCTCCCCATCATCAACCGCCTGATTCTGCAAAGCCCGGACAGCCGCTCG GAACTTGCCGCCTTTGCAGGCΆAAACACTGACCCTGAACATTGCCGGGCTGAAΆCTGGCG GGACGCATCACGGAAGACGGTTTGCTCTCGGCGGGAΆACGGCTTTGCAGACACCGAAΆTT ACCTTCCGCAΆCAGCGCGGTACAGAΆAATCCTCCAAGGAGGCGAACCCGGGGCGGGCGAC ΆTCGGGCTCGAAGGCGACCTCATCCTCGGCATCGCGGTACTGTCCCTGCTCGGCAGCCTG CGTTCCCGCGCATCGGACGAATTGGCACGGATTTTCGGCACGCAGGCAGACATCGGCAGC CGTGCCGCCGACATCGGACACGGCATCAAΆCAAATCGGCAGGAACATCGCCGAACAΆATC GGCGGATTTTCCCGCGAATCCGAGTCCGCAAACATCGGCAACGAAGCCCTTGCCGACTGC CTCGACGAAATAAGCAGACTGCGCGACGGCGTGGAACGCCTCAACGAACGCCTCGACCGG CTCGAACGCGACATTTGGATAGACTAA
NMB0808 Protein sequence
MSALLPIINRLILQSPDSRSELAAFAGKTLTLNIAGLKLAGRITEDGLLSAGNGFADTEI TFRNSAVQKILQGGEPGAGDIGLEGDLILGIAVLSLLGSLRSRASDELARIFGTQADIGS RAADIGHGIKQIGRNIAEQIGGFSRESESANIGNEALADCLDEISRLRDGVERLNERLDR LERDIWID
NMB0774 upp DNA sequence
ATGAACGTTAATGTTATCAACCATCCGCTCGTCCGCCACAAATTAACCCTGATGAGGGAG GCGGATTGCAGCACCTACAAATTCCGGACGCTTGCCACCGAGCTGGCGCGCCTGATGGCA TACGAGGCAAGCCGTGATTTTGAAATCGAAAAATACCTTATCGACGGATGGTGCGGTCAG ATTGAAGGCGACCGCATCAAGGGCAAAACATTGACCGTCGTTCCCATACTGCGTGCAGGT TTGGGTATGCTTGACGGTGTGCTCGACCTGATTCCGACTGCCAAAATCAGTGTAGTCGGA CTGCAGCGCGACGAAGAAACGCTGAAGCCTATTTCCTATTTTGAGAAATTTGTGGACAGT ATGGACGAACGTCCGGCTTTGATTATCGATCCTATGCTGGCGACΆGGCGGTTCGATGGTT GCCACCATCGACCTTTTGAAAGCCAAGGGCTGCAAAAATATCAAGGCACTGGTGCTGGTT GCCGCGCCCGAGGGTGTGAAGGCGGTCAACGACGCGCACCCTGACGTTACGATTTACACC GCCGCGCTCGACAGCCACTTGAACGAGAACGGCTACATCATCCCCGGCTTGGGCGATGCG GGCGACAAGATTTTCGGCACGCGCTAA
NMB0774 Protein sequence
MNVNVINHPLVRHKLTLMREADCSTYKFRTLATELARLMAYEASRDFEΪERYLIDGWCGQ IEGDRIKGKTLTWPILRAGLGMLDGVLDLIPTAKISVVGLQRDEETLKPISYFEKFVDS MDERPALI IDPMLATGGSMVATIDLLKAKGCKNIKALVLVAAPEGVKAVNDAHPDVTIYT AALDSHLNENGYIIPGLGDAGDKIFGTR
NMA0078 putative integral membrance protein DNA sequence TTGGCGTTTACTTTAATGCGTCGCGCCATGATACGTAAAATGCCCTATACGGAAGATATG
CGCCCAGGCGATACCGCTAATCCTTATGGTGCGTCCAAAGCGATGGTGGAΆCGGATGTTA ACCGACATCCAAAAAGCCGATCCGCGCTGGAGCATGATTTTGTTGCGTTATTTCAATCCG ATTGGCGCGCATGAAAGCGGCTTGATTGGCGAGCAGCCAAACGGCATCCCGAATAATTTG TTGCCTTATΆTCTGCCAAGTGGCGGCAGGCAAACTGCCGCAATTGGCGGTATTTGGCGAT GACTACCCTACCCCCGACGGCACGGGGATGCGTGACTATATTCATGTGATGGATTTGGCA GAAGGCCATGTCGCGGCTATGCAGGCAAAAAGTAATGTAGCAGGCACGCATTTGCTGAAC TTAGGCTCCGGCCGCGCTTCTTCGGTGTTGGAAATCATCCGCGCATTTGAAGCAGCTTCG GGTTTGACGATTCCGTATGAAGTCAAACCGCGCCGTGCCGGTGATTTGGCGTGCTTCTAT GCCGACCCTTCCTATACAAAGGCGCAΆATCGGCTGGCΆAΆCCCAGCGTGATTTAACCCAΆ ΆTGATGGAAGACTCATGGCGCTGGGTGAGTΆATAATCCGAΆTGGCTACGACGATTAA
NMA0078 Protein sequence
MAFTLMRRAMIRKMPYTEDMRPGDTANPYGASKAMVERMLTDIQKADPRWSMILLRYFNP IGAHESGLIGEQPNGIPNNLLPYICQVAAGKLPQLAVFGDDYPTPDGTGMRDYIHVMDLA EGHVAAMQAKSNVAGTHLLNLGSGRASSVLEIIRAFEAASGLTIPYEVKPRRAGDLACFY ADPSYTKAQIGWQTQRDLTQMMEDSWRWVSNNPNGYDD
NMB0337 Branched-chain amino acid aminotransferaseDNA sequence
ATGAGCAGACCCGTACCCGCCGTATTCGGCAGCGTTTTTCACAGTCAAATGCCCGTCCTC GCCTACCGCGAAGGCAAATGGCAGCCGACCGAATGGCAATCTTCCCAAGACCTCTCCCTC GCACCGGGCGCGCACGCCCTGCACTACGGCAGCGAATGTTTCGAGGGΆCTGAAAGCCTTC CGTCAGGCAGACGGCAAAATCGTGCTGTTCCGTCCGACTGCCAATATCGCGCGTATGCGG CAAAGTGCGGACATTTTGCACCTGCCGCGCCCCGAAACCGAAGCTTATCTTGACGCGCTA ATCAAATTGGTCAAACGTGCCGCCGATGAAATTCCCGATGCGCCTGCCGCCCTGTACCTG CGTCCGACCTTAATCGGTACCGATCCCGTTATCGGCAAGGCCGGTTCTCCTTCCGAAACC GCCCTGCTGTATATTTTGGCTTCCCCCGTCGGCGACTATTTCAAAGTCGGATCGCCCGTC AAAATTTTGGTGGAAACCGAACACATCCGCTGCGCCCCGCATATGGGCCGCGTCAAATGC GGCGGCAACTACGCTTCCGCCATGCACTGGGTGCTGAΆGGCGAΆAGCCGAATATGGCGCA AATCAAGTCCTGTTCTGCCCGAACGGCGACGTGCAGGAAACCGGCGCGTCCAACTTTATC CTGATTAACGGCGATGAAATCATTACCAAACCGCTGACCGACGAGTTTTTGCACGGCGTA ACCCGCGATTCCGTACTGACGGTTGCCAAAGATTTGGGCTATACCGTCAGCGAACGCAAT TTCΆCGGTTGACGAACTCAAAGCTGCGGTGGAAAACGGTGCGGAAGCCΆTTTTGACCGGT ACGGCAGCCGTCATCTCGCCCGTTACTTCCTTCGTCATCGGCGGCAΆAGAΆATCGAAGTG AAAAGCCAAGAACGCGGCTATGCCATCCGTAAGGCGATTACCGACATCCAGTATGGTTTG GCGGAAGACAAATACGGCTGGCTGGTTGAAGTGTGCTGA
NMB0337 Protein sequence
MSRPVPAVFGSVFHSQMPVLAYREGKWQPTEWQSSQDLSLAPGAHALHYGSECFEGLKAF RQADGKIVLFRPTANIARMRQSADILHLPRPETEAYLDALIKLVKRAADEIPDAPAALYL RPTLIGTDPVIGKAGSPSETALLYILASPVGDYFKVGSPVKILVETEHIRCAPHMGRVKC GGNYASAMHWVLKAKAEYGANQVLFCPNGDVQETGASNFILINGDEIITKPLTDEFLHGV TRDSVLTVAKDLGYTVSERNFTVDELKAAVENGAEAILTGTAAVISPVTSFVIGGKEIEV KSQERGYAIRKAITDIQYGLAEDKYGWLVEVC NMB0191 ParA family protein DNA sequence
ATGAGTGCGAACATCCTTGCCATCGCCAATCAGAAGGGCGGTGTGGGCAAAACGACGACG ACGGTAAATTTGGCGGCTTCGCTGGCATCGCGCGGCAAACGCGTGCTGGTGGTCGATTTG GATCCGCAGGGCAATGCGΆCGACGGGCAGCGGCATCGACAAGGCGGGTTTGCΆGTCCGGC GTTTATCAGGTCTTATTGGGCGATGCGGACGTGCAGTCGGCGGCGGTACGCAGCAΆΆGAG GGCGGATACGCTGTGTTGGGTGCGAACCGCGCGCTGGCCGGCGCGGAAATCGAACTGGTG CAGGAAΆTCGCCCGGGAAGTGCGTTTGAΆΆAACGCGCTCAΆGGCΆGTGGAAGAAGATTΆC GACTTTΆTCCTGATCGACTGCCCGCCTTCGCTGACGCTGTTGACGCTTAACGGGCTGGTG GCGGCGGGCGGCGTGATTGTGCCGATGTTGTGCGAΆTATTACGCGCTGGAΆGGGATTTCC GΆTTTGATTGCGACCGTGCGCΆΆAΆTCCGTCΆGGCGGTCΆATCCCGATTTGGACATCACG GGCATCGTGCGCACGATGTACGACAGCCGCAGCAGGCTGGTTGCCGAAGTCAGCGAACAG TTGCGCAGCCATTTCGGGGATTTGCTTTTTGAAACCGTCATCCCGCGCAATATCCGCCTT GCGGAΆGCGCCGΆGCCACGGTATGCCGGTGATGGCTTACGACGCGCAGGCAAΆGGGTACC AAGGCGTATCTTGCCTTGGCGGACGAGCTGGCGGCGAGGGTGTCGGGGAAATAG
NMB0191 Protein sequence MSANILAIANQKGGVGKTTTTVNLAASLASRGKRVLWDLDPQGNATTGSGIDKAGLQSG VYQVLLGDΆDVQSAAVRSKEGGYAVLGANRALAGAEIELVQEIAREVRLKNALKAVEEDY DFILIDCPPSLTLLTLNGLVAAGGVIVPMLCEYYALEGISDLIATVRKIRQAVNPDLDIT GIVRTMYDSRSRLVAEVSEQLRSHFGDLLFETVIPRNIRLAEAPSHGMPVMAYDAQAKGT KAYLALADELAARVSGK
NMB1710 Glutamate dehydrogenase(gdhA) DNA sequence ATGACTGACCTGAACACCCTGTTTGCCAACCTCAAACAACGCAATCCCAATCAGGAGCCG TTCCATCAGGCGGTTGAAGAAGTCTTCATGAGTCTCGATCCGTTTTTGGCAΆAAAATCCG AAATACACCCΆGCAAAGCCTGCTGGAACGCATCGTCGAACCCGAACGCGTCGTGATGTTC CGCGTAACCTGGCAGGACGATAAAGGGCAAGTCCAAGTCAACCGGGGCTACCGCGTGCAA ATGAGTTCCGCCATCGGTCCTTACAAAGGCGGCCTGCGCTTCCATCCGACCGTCGATTTG GGCGTATTGΆAΆTTCCTCGCTTTTGΆACAAGTGTTCAΆAAΆCGCCTTGACCACCCTGCCT ATGGGCGGCGGCAAAGGCGGTTCCGACTTCGACCCCAAAGGCAAATCCGATGCCGAAGTA ATGCGCTTCTGCCAAGCCTTTATGACCGAACTCTACCGCCACATCGGCGCGGACACCGAT GTTCCGGCCGGCGACATCGGCGTAGGCGGGCGCGAAATCGGCTACCTGTTCGGACAATAC
AAAAAAATCCGCAACGAGTTTTCTTCCGTCCTGACCGGCAAAGGTTTGGAATGGGGCGGC
AGCCTCATCCGTCCCGAΆGCGACCGGCTACGGCTGCGTCTATTTCGCCCAAGCGΆTGCTG CAAACCCGCAACGATAGTTTTGAAGGCΆAACGCGTCCTGATTTCCGGCTCCGGCAATGTG GCGCAATACGCCGCCGAAAAΆGCCATCCAΆCTGGGTGCGAΆΆGTACTGACCGTTTCCGAC TCCAACGGCTTCGTCCTCTTCCCCGACAGCGGTATGACCGAAGCGCAACTCGCCGCCTTG ATCGAATTGAAAGAAGTCCGCCGCGAACGCGTTGCCACCTACGCCAAAGAGCAAGGTCTG CΆATACTTTGAAAAACAAAAACCGTGGGGCGTCGCCGCCGAΆATCGCCCTGCCCTGCGCG ACCCAGAACGAATTGGACGAAGAΆGCCGCCAAAACCCTGTTGGCΆAACGGCTGCTACGTC GTTGCCGAAGGTGCGAATATGCCGTCGACTTTGGGCGCGGTCGAGCAATTTATCAAAGCC GGCATCCTCTACGCCCCGGGAAAAGCCTCCAATGCCGGCGGCGTGGCAACTTCAGGTTTG GAAΆTGAGCCAAAACGCCATCCGCCTGTCTTGGACTCGTGAΆGAAGTCGACCAACGCCTG TTCGGCATCATGCAAAGCATCCACGAATCCTGTCTGAAATACGGCAAAGTCGGCGACACA GTAΆACTACGTCAATGGTGCGAACATTGCCGGTTTCGTCAAAGTTGCCGATGCGATGCTG GCGCAAGGCTTCTAA
NMB1710 Protein sequence
MTDLNTLFANLKQRNPNQEPFHQAVEEVFMSLDPFLAKNPKYTQQSLLERIVEPERWMF RVTWQDDKGQVQVNRGYRVQMSSAIGPYKGGLRFHPTVDLGVLKFLAFEQVFKNALTTLP MGGGKGGSDFDPKGKSDAEVMRFCQΆFMTELYRHIGADTDVPAGDIGVGGREIGYLFGQY KKIRNEFSSVLTGKGLEWGGSLIRPEATGYGCVYFAQAMLQTRNDSFEGKRVLISGSGNV AQYAAEKAIQLGAKVLTVSDSNGFVLFPDSGMTEAQLAALIELKEVRRERVATYAKEQGL QYFEKQKPWGVAAEIALPCATQNELDEEAAKTLLANGCYWAEGANMPSTLGAVEQFIKA GILYAPGKASNAGGVATSGLEMSQNAIRLSWTREEVDQRLFGIMQSIHESCLKYGKVGDT VNYVNGANIAGFVKVADAMLAQGF
NMB0062 Glucose- 1 -phosphate thymidylytransferase(rfbA-l) DNA sequence
ATGAAAGGCATCATACTGGCAGGCGGCAGCGGCACGCGCCTCTACCCCATCACGCGCGGC GTATCCAAACAGCTCCTGCCCGTGTACGACAAACCGATGATTTATTACCCCTTGTCGGTT TTGATGCTGGCGGGAATCCGCGATATTTTGGTGATTΆCCGCGCCΓGAAGACAΆCGCCTCT TTCAΆACGCCTGCTTGGCGACGGCAGCGATTTCGGCATTTCCATCAGTTATGCCGTGCAA CCCΆGTCCGGACGGCTTGGCACAGGCATTTΆTCATCGGCGAAGAΆTTTATCGGCΆACGAC AATGTTTGCTTGGTTTTGGGCGACAATATTTTTTACGGTCAGTCGTTTACGCAAACATTG AAACAGGCGGCAGCGCAAACGCACGGCGCAACCGTGTTTGCTTATCAGGTCAAAAACCCC GAΆCGTTTCGGCGTGGTTGAATTTAACGAAAΆCTTCCGCGCCGTTTCCATCGAAGAAAAA CCGCAACGGCCCAAATCCGATTGGGCGGTAACCGGCTTGTATTTCTACGACAACCGCGCC GTCGAGTTCGCCAAACAGCTCAAACCGTCCGCACGCGGCGAATTGGAAATTACCGΆCCTC AACCGGATGTATTTGGAAGACGGCTCGCTCTCCGTTCAAATATTGGGACGCGGTTTCGCG TGGCTGGACACCGGCACCCACGAGAGCCTGCACGAAGCCGCTTCATTCGTCCAAACCGTG CAΆΆATATCCAAAACCTGCACATCGCCTGCCTCGAAGAΆΆTCGCTTGGCGCAACGGTTGG CTTTCCGATGAAAAACTGGAAGAATTGGCGCGCCCGATGGCGAAAAACCAATACGGCCAA TATTTGCTGCGCCTGTTGAAΆAAATAΆ NMB0062 Protein sequence
MKGIILAGGSGTRLYPITRGVSKQLLPVYDKPMIYYPLSVLMLAGIRDILVITAPEDNAS FKRLLGDGSDFGISISYAVQPSPDGLAQAFIIGEEFIGNDNVCLVLGDNIFYGQSFTQTL KQAAΆQTHGATVFAYQVKNPERFGVVEFNENFRAVSIEEKPQRPKSDWAVTGLYFYDNRΆ VEFAKQLKPSARGELEITDLNRMYLEDGSLSVQILGRGFAWLDTGTHESLHEAASFVQTV QNIQNLHIACLEEIAWRNGWLSDEKLEELARPMAKNQYGQYLLRLLKK
NMB1583 Imidazoleglycerol-phosphate dehydratase(hisB) DNA sequence
ATGAATTTGACTAAAΆCACAACGCCAACTGCACAACTTTCTGACCCTCGCCCAΆGAAGCA GGTTCGCTGTCCAAGCTCGCCAAACTCTGCGGCTACCGTACCCCCGTCGCACTCTACAΆA CTCAAACAACGCCTTGAAAAGCAGGCAGAAGACCCAGATGCACGCGGCATCCGTCCCAGC CTGATGGCAAΆACTCGAAAAACACACCGGCAΆACCCAΆAGGCTGGCTCGACAGAΆAACAC CGCGAACGCACTGTCCCCGAAΆCCGCCGCAGAAAGCACCGGAΆCTGCCGAAΆCCCAAATT GCCGAAACCGCATCTGCTGCCGGCTGCCGCAGCGTTACCGTCAACCGCAATACCTGCGAA ACCCAAATCACCGTCTCCATCAACCTCGACGGCAGCGGCAAAAGCAGGCTGGATACCGGC GTACCCTTCCTCGAACACATGATCGATCAAATCGCCCGCCACGGCATGATTGACATCGAC ATCAGCTGCAAAGGCGACCTGCACATCGACGACCACCACACCGCCGAAGACATCGGCATC ACACTCGGACΆAGCAATCCGGCAGGCACTCGGCGACAAAAAAGGCATCCGCCGTTACGGA CATTCCTACGTCCCGCTCGACGAAGCCCTCAGCCGCGTCGTCATCGACCTTTCCGGCCGC CCCGGACTCGTGTACAACATCGAATTTACCCGCGCACTAATCGGACGTTTCGATGTCGΆT TTGTTTGAAGAATTTTTCCACGGCATCGTCAACCACAGTATGATGACCCTGCACATCGAC AΆCCTCAGCGGCAAAAACGCCCACCATCAGGCGGAAACCGTATTCAAAGCCTTCGGGCGC GCCCTGCGTATGGCAGTCGAACACGACCCGCGCATGGCAGGACAGACCCCCTCGACCAAA GGCACGCTGACCGCATAA NMB1583 Proteinsequence
MNLTKTQRQLHNFLTLAQEAGSLSKLAKLCGYRTPVALYKLKQRLEKQAEDPDARGIRPS LMAKLEKHTGKPKGWLDRKHRERTVPETAAESTGTAETQIAETASAAGCRSVTVNRNTCE TQITVSINLDGSGKSRLDTGVPFLEHMIDQIARHGMIDIDISCKGDLHIDDHHTAEDIGI TLGQAIRQALGDKKGIRRYGHSYVPLDEALSRVVIDLSGRPGLVYNIEFTRΆLIGRFDVD LFEEFFHGIVNHSMMTLHIDNLSGKNAHHQAETVFKAFGRALRMAVEHDPRMAGQTPSTK
GTLTA
The following additional antigens were identified using essentially the methodology described above:
NMB 1333 Nucleic acid sequence
ATGCGCTACAAACCCCTTCTGCTTGCCCTGATGCTCGTTTTTTCCACGCCCGCCGTTGCC
GCCCACGACGCGGCACACAACCGTTCCGCCGAAGTGAAAAAACAGACGAΆGAACAAAAAA GAACAGCCCGAAGCGGCGGAAGGCAAAAAAGAAAAAGGCAAAAATGGCGCAGTGAAAGAT AAAA-AAACAGGCGGCAAAGAGGCGGCAAAAGAGGGCAAAGAGTCCAAAAAAACCGCCAAA
AACCGCAAAGAAGCAGAGAAGGAGGCGACATCCAGGCAGTCTGCGCGCAAAGGACGCGAA GGGGATAAGAAATCGAAGGCGGAΆCΆCAAAAAGGCACATGGCAΆGCCCGTGTCCGGATCC AAAGAAAAAAACGCAAAAACACAGCCTGAAAACAAACAAGGCAAAAAAGAGGCAAAAGGA CAGGGCAATCCGCGCAAGGGCGGCAAGGCGGAAAAAGACACTGTTTCTGCAAATAAAAAA GTCCGTTCCGACAΆGAACGGCAAΆGCAGTGAAΆCAGGACAAAΆAATACAGGGAAGAGAAA AATGCCAAAACCGATTCCGACGAATTGAAAGCCGCCGTTGCCGCTGCCACCAATGATGTC GAAAACAAAAAAGCCCTGCTCAAACAAAGCGAAGGAATGCTGCTTCATGTCAGCAATTCC CTCAAACAGCTTCAGGAAGAGCGTATCCGCCAAGAGCGTATCCGTCAGGCGCGCGGCAAC CTTGCTTCCGTCAACCGCAAACAGCGCGAGGCTTGGGACAAGTTCCAAAAACTCAATACC GAGCTGAACCGTTTGAAAACGGAAGTCGCCGCTACGAAΆGCGCAGATTTCCCGTTTCGTA TCGGGGAACTATAAAAACAGCCAGCCGAATGCGGTTGCCCTGTTCCTGAAAAACGCCGAA CCGGGTCAGAAAAACCGCTTTTTGCGTTATACGCGTTATGTAAACGCCTCCAATCGGGAA GTTGTCAAGGATTTGGAAAAACAGCAGAAGGCTTTGGCGGTACAAGAGCAGAAΆATCAΆC AATGAGCTTGCCCGTTTGAΆGΆΆAATTCAGGCAAACGTGCAATCTCTGCTGAΆΆAAACAG GGTGTAACCGATGCGGCGGΆACAGACGGAAAGCCGCAGACAGAATGCCAAAATCGCCAAA GATGCCCGAAΆACTGCTGGAACAGAAAGGGAACGAGCAGCAGCTGAACAAGCTCTTGAGC AATTTGGAGAAGAAAAAGGCCGAACACCGCATTCAGGATGCGGAAGCAAAAΆGAAAATTG GCTGAΆGCCAGACTGGCGGCAGCCGAAAAAGCCAGAAAAGAAGCGGCGCΆGCAGAAGGCT GAAGCACGACGTGCGGAAATGTCCAACCTGACCGCCGAAGACAGGAACATCCAAGCGCCT TCGGTTATGGGTATCGGCAGTGCCGACGGTTTCΆGCCGCATGCAAGGACGTTTGAAAAAA CCGGTTGΆCGGTGTGCCGACCGGACTTTTCGGGCAGAACCGGAGCGGCGGCGATATTTGG AAAGGCGTGTTCTATTCCACTGCACCGGCΆΆCGGTTGAAAGCATTGCGCCGGGAACGGTA AGCTATGCGGΆCGAGTTGGACGGCTACGGCAAΆGTGGTCGTGGTCGATCACGGCGAGAAC TACATCAGCATCTATGCCGGTTTGAGCGAAATTTCCGTCGGCAAGGGTTATATGGTCGCG GCΆGGAAGCAAAATCGGCTCGΆGCGGGTCGCTGCCGGACGGGGAAGAGGGGCTTTACCTG CAAATACGTTATCAAGGTCAGGTATTGAΆCCCTTCGAGCTGGATACGTTGA
NMB1333 Amino acid sequence MRYKPLLLALMLVFSTPAVAAHDAAHNRSAEVKKQTKNKKEQPEAAEGKKEKGKNGAVKD KKTGGKEAAKEGKESKKTAKNRKEAEKEATSRQSARKGREGDKKSKAEHKKAHGKPVSGS KEKNAKTQPENKQGKKEAKGQGNPRKGGKΆEKDTVSANKKVRSDKNGKAVKQDKKYREEK NΆKTDSDELKAAVAAATNDVENKKALLKQSEGMLLHVSNSLKQLQEERIRQERIRQARGN LASVNRKQREAWDKFQKLNTELNRLKTEVAATKAQISRFVSGNYKNSQPNAVALFLKNAE PGQKNRFLRYTRYVNASNREWKDLEKQQKALAVQEQKINNELARLKKIQANVQSLLKKQ GVTDAAEQTESRRQNAKIAKDARKLLEQKGNEQQLNKLLSNLEKKKAEHRIQDAEAKRKL AEARLAAAEKARKEAAQQKΆEARRAEMSNLTAEDRNIQAPSVMGIGSADGFSRMQGRLKK PVDGVPTGLFGQNRSGGDIWKGVFYSTAPATVESIAPGTVSYADELDGYGKWWDHGEN YISIYAGLSEISVGKGYMVAAGSKIGSSGSLPDGEEGLYLQIRYQGQVLNPSSWIR
NMB0377 Nucleicacid sequence
ATGGCGTTTTGCACCAGTTTGGGAGTGATGATGGAAACACAGCTTTACATCGGCATCATG TCGGGAACCAGCATGGACGGGGCGGATGCCGTACTGATACGGATGGACGGCGGCAAATGG CTGGGCGCGGAAGGGCACGCCTTTACCCCCTACCCCGGCAGGTTACGCCGCCAATTGCTG GATTTGCAGGACACAGGCGCAGACGAACTGCACCGCAGCAGGATTTTGTCGCAAGAACTC AGCCGCCTATATGCGCAAACCGCCGCCGAACTGCTGTGCAGTCAAΆACCTCGCACCGTCC GACATTACCGCCCTCGGCTGCCACGGGCAAΆCCGTCCGACACGCGCCGGAACACGGTTAC AGCATACAGCTTGCCGATTTGCCGCTGCTGGCGGAACGGACGCGGATTTTTACCGTCGGC GACTTCCGCAGCCGCGACCTTGCGGCCGGCGGACAAGGCGCGCCACTCGTCCCCGCCTTT CACGAAGCCCTGTTCCGCGΆCAACAGGGAAACACGCGCGGTACTGAΆCATCGGCGGGATT GCCAACATCAGCGTACTCCCCCCCGACGCACCCGCCTTCGGCTTCGACACAGGGCCGGGC AATATGCTGATGGACGCGTGGACGCAGGCACACTGGCAGCTTCCTTACGACAAAAACGGT GCAAAGGCGGCACAAGGCAACATATTGCCGCAACTGCTCGACAGGCTGCTCGCCCACCCG TATTTCGCACAACCCCACCCTAAAAGCACGGGGCGCGAACTGTTTGCCCTAAATTGGCTC GAAACCTACCTTGACGGCGGCGAAAACCGATACGACGTATTGCGGACGCTTTCCCGTTTT ACCGCGCAAACCGTTTGCGACGCCGTCTCACACGCAGCGGCAGATGCCCGTCAAATGTAC ATTTGCGGCGGCGGCATCCGCAATCCTGTTTTAATGGCGGATTTGGCAGAATGTTTCGGC ACACGCGTTTCCCTGCACAGCACCGCCGACCTGAACCTCGATCCGCAATGGGTGGAAGCC
GCCGCATTTGCGTGGTTGGCGGCGTGTTGGATTAATCGCATTCCCGGTAGTCCGCACAAA
GCAACCGGCGCATCCAAACCGTGTATTCTGGGCGCGGGATATTATTATTGA NMB 0377 Amino acid sequence
MAFCTSLGVMMETQLYIGIMSGTSMDGADAVLIRMDGGKWLGAEGHAFTPYPGRLRRQLL DLQDTGADELHRSRILSQELSRLYAQTAAELLCSQNLAPSDITALGCHGQTVRHAPEHGY SIQLADLPLLAERTRIFTVGDFRSRDLAAGGQGAPLVPAFHEALFRDNRETRAVLNIGGI ANISVLPPDAPAFGFDTGPGNMLMDAWTQAHWQLPYDKNGAKAAQGNILPQLLDRLLAHP YFAQPHPKSTGRELFALNWLETYLDGGENRYDVLRTLSRFTAQTVCDAVSHAAADARQMY ICGGGIRNPVLMADLAECFGTRVSLHSTADLNLDPQWVEAAAFAWLAACWINRIPGSPHK ATGASKPCILGAGYYY
NMB0264Nucleic acid sequence ATGTTGAACAAAATATTTTCCTGGTTCGAGTCCCGAATCGACCCTTATCCCGAAGCCGCC CCGΆAΆΆCGCCAGAAAAΆGGCTTGTGGCGGTTTGTCTGGAGCAGCATGGCCGGCGTGCGG AAATGGATAGCCGCCCTGGCTGCGCTGACCGCCGGCATCGGCATTATGGAΆGCCCTGGTT TTTCAATTTATGGGCAAAATCGTGGAGTGGCTCGGCAAΆTACGCGCCCGCCGΆACTGTTT GCCGAAAAAAGTTGGGAACTGGCGGCAATGGCGGCGATGATGGTATTTTCGGTTGCGTGG GCGTTTGCCGCGTCCAACGTGCGCCTGCAAACCCTTCAGGGCGTGTTCCCCATGCGCCTG CGCTGGAACTTCCACCGCCTGATGCTGAACCΆAAGCCTCGGTTTTTATCAGGACGAATTT GCCGGACGCGTGTCCGCCΆAAGTCΆTGCAGACCGCGCTGGCGTTGCGCGACGCGGTGΆTG
ACGGTTGCCGATATGGTCGTTTATGTGTCGGTGTATTTCATTACCTCCGGCGTGATTCTC
GCCTCGCTCGACTCATGGCTGCTGCTGCCCTTTATCGGCTGGATTGTCGGTTTCGCTTCG GTGATGCGCCTGCTGATTCCCAAATTGGGGCAAACCGCCGCATGGCAGGCGGATGCCCGC TCGCTGATGACCGGCCGCATTACCGATGCCTATTCCAATATCGCCACCGTCAAACTCTTC TCCCACGGCGCGCGTGAAGCCGCCTATGCCAAGCAGTCGATGGAAGAATTTATGGTTACG GTGCGCGCCCΆAATGCGGCTGGCGΆCGCTGCTGCATTCGTGCAGCTTCATCGTCAACACC TCCCTGACCCTCTCCACCGCCGCACTGGGCATCTGGCTCTGGCΆCΆΆCGGGCAGGTCGGC GTGGGCGCGGTTGCTACAGCCACCGCCATGGCGTTGCGCGTCAACGGTTTGTCGCAATAC ATTATGTGGGAATCCGCGCGGCTGTTTGAAAACATCGGCACCGTCGGCGACGGCATGGCA ACCCTGTCCAAACCGCACACCATCCTCGACAAGCCCCGGGCACTGCCGCTGAACGTGCCG CAΆGGCGCAATCAAΆTTTGAACACGTCGATTTCTCCTACGAAGCGGGCAΆACCGCTGCTC AACGGCTTCAACCTCACCATCCGCCCGGGCGAΆΆΆAGTCGGCTTGATCGGACGCAGCGGC GCGGGCAAATCCACCATCGTCAACCTGCTTTTGCGCTTCTACGAACCGCAAAGCGGCACG GTTTCGATCGACGGGCΆGGACATAAGCGGCGTTACCCAAGAATCTTTACGCGCCCAAATC GGTTTGGTCACGCAAGATACCTCGCTGCTGCACCGTTCCGTGCGCGACAΆCATTATTTAC GGCCGCCCCGACGCGACCGATGCCGAAATGGTTTCTGCCGCCGAACGCGCCGAAGCCGCC GGCTTCATCCCCGACCTTTCCGATGCCAAAGGGCGGCGCGGCTACGACGCACACGTCGGC GAACGCGGCGTGAAACTCTCCGGCGGGCAACGCCAGCGCATCGCCATCGCCCGCGTGATG
CTCAAAGACGCΆCCGATTCTTCTTTTGGACGAAGCCACCAGCGCGCTCGATTCCGAAGTC
GAAGCCGCCATCCAAGAAΆGCCTCGACAAAATGATGGACGGCAAAACCGTCATCGCCΆTC
■ GCCCACCGCCTCTCCACCΆTCGCCGCAΆTGGACAGGCTCGTCGTCCTCGΆCAAΆGGCCGC
ATCATCGAAGAAGGCACACACGCCGAACTCCTCGAAAAACGCGGGCTTTACGCCAAACTC • TGGGCGCACCAGAGCGGCGGCTTCCTCAACGAACACGTCGAGTGGCAGCACGACTGA
NMB0264 Amino acid sequence
MLNKIFSWFESRIDPYPEAAPKTPEKGLWRFVWSSMAGVRKWIAALAALTAGIGIMEALV FQFMGKIVEWLGKYAPAELFAEKSWELAAMAAMMVFSVAWAFAASNVRLQTLQGVFPMRL RWNFHRLMLNQSLGFYQDEFAGRVSAKVMQTALALRDAVMTVADMVVYVSVYFITSGVIL ΆSLDSWLLLPFIGWIVGFASVMRLLIPKLGQTAAWQADARSLMTGRITDAYSNIΆTVKLF SHGAREAAYAKQSMEEFMVTVRAQMRLATLLHSCSFIVNTSLTLSTAALGIWLWHNGQVG VGAVATATAMALRVNGLSQYIMWESARLFENIGTVGDGMATLSKPHTILDKPRALPLNVP QGAIKFEHVDFSYEAGKPLLNGFNLTIRPGEKVGLIGRSGAGKSTIVNLLLRFYEPQSGT VSIDGQDISGVTQESLRAQIGLVTQDTSLLHRSVRDNIIYGRPDATDAEMVSAAERAEAA GFIPDLSDAKGRRGYDAHVGERGVKLSGGQRQRIAIARVMLKDAPILLLDEATSALDSEV EAAIQESLDKMMDGKTVIAIAHRLSTIAMDRLWLDKGRIIEEGTHAELLEKRGLYAKL WAHQSGGFLNEHVEWQHD NMB1036 Nucleic acid sequence
ATGACAGCACAAACCCTCTACGΆCAAACTTTGGAACAGCCACGTCGTCCGCGAAGAAGAΆ GACGGCACCGTCCTGCTCTACATCGACCGCCATTTGGTGCACGAAGTTACCAGCCCTCAG GCATTTGAAGGCTTGAAAATGGCGGGGCGCAAGCTGTGGCGCATCGACAGCGTCGTCTCC ACCGCCGΆCCACAACACCCCGACCGGCGATTGGGACAAAGGCATCCAAGACCCGATTTCC AAGCTGCAAGTCGATACTTTGGACAAAAACATTAAAGAGTTTGGCGCACTCGCCTATTTT CCGTTTATGGACΆAAGGTCAGGGCΆTCGTACACGTTATGGGCCCCGAACAAGGCGCGACC CTGCCCGGTATGΆCCGTCGTCTGCGGCGACTCGCACACTTCCACCCACGGCGCATTCGGC GCACTGGCGCACGGCATCGGCACTTCCGAAGTCGAGCACACCATGGCGACCCAATGTATT ACCGCGAAAAAATCCAAATCCATGCTGATTTCCGTTGACGGCAAATTAAAAGCGGGCGTT ACCGCCAAAGACGTGGCGCTCTACATCATCGGGCAAATCGGCACGGCAGGCGGTACAGGC TACGCCATCGAGTTTGGCGGCGAAGCCATCCGCAGCCTTTCTATGGAAAGCCGCΆTGACT TTATGCΆATATGGCGATTGΆGGCΆGGCGCGCGCTCAGGCATGGTTGCCGTCGACCAAΆCC ACCATCGACTACGTAAAAGATAAACCCTTCGCACCCGAAGGCGAAGCGTGGGACAAAGCC GTCGAGTACTGGCGTACGCTGGTGTCTGACGAAGGTGCGGTATTCGACAAΆGΆATACCGT TTCAACGCCGAAGACATCGAACCGCAAGTCACTTGGGGTACCTCGCCTGAAATGGTTTTA GACATCAGCAGCAAAGTGCCGΆATCCTGCCGAAGAAΆCCGATCCGGTCAAACGCΆGCGGT ATGGAACGCGCCCTTGAATΆCATGGGCTTGGAAGCCGGTACGCCATTAAACGAAΆTCCCC GTCGACATCGTATTCATCGGCTCTTGCACCAACAGCCGCATCGAAGACTTGCGCGAAGCC GCCGCCATCGCCAAΆGACCGCAAAAAAGCCGCCAACGTACAGCGCGTGTTAATCGTCCCC GGCTCCGGTTTGGTTAΆAGAACAΆGCCGAAΆAAGAAGGCTTGGACAAAATTTTCΆTCGAΆ GCCGGTTTTGAATGGCGCGAACCGGGCTGTTCGATGTGTCTCGCCATGAACGCCGACCGC CTGACCCCGGGGCAACGCTGCGCCTCCACCTCCAACCGTAACTTTGAAGGCCGTCAAGGC AACGGCGGACGTACCCACCTCGTCAGCCCCGCTATGGCAGCAGCCGCCGCCGTTACCGGC CGCTTTACCGACATCCGCATGATGGCGTAA
NMB1036 Amino acid sequence MTAQTLYDKLWNSHWREEEDGTVLLYIDRHLVHEVTSPQAFEGLKMAGRKLWRIDSVVS TADHNTPTGDWDKGIQDPISKLQVDTLDKNIKEFGALAYFPFMDKGQGIVHVMGPEQGAT LPGMTWCGDSHTSTHGAFGALAHGIGTSEVEHTMΆTQCITΆKKSKSMLISVDGKLKAGV TAKDVALYIIGQIGTAGGTGYAIEFGGEAIRSLSMESRMTLCNMAIEAGARSGMVAVDQT TIDYVKDKPFAPEGEAWDKAVEYWRTLVSDEGAVFDKEYRFNAEDIEPQVTWGTSPEMVL DISSKVPNPAEETDPVKRSGMERALEYMGLEAGTPLNEIPVDIVFIGSCTNSRIEDLREA AAIAKDRKKAANVQRVLIVPGSGLVKEQAEKEGLDKIFIEAGFEWREPGCSMCLAMNADR LTPGQRCASTSNRNFEGRQGNGGRTHLVSPAMAAAAAVTGRFTDIRMMA
NMB1176 Nucleic acid sequence ATGAAAGACAAGCACGATTCTTCCGCCATGCGGCTGGACAAATGGCTTTGGGCGGCACGT TTTTTCAΆGACCCGTTCCCTTGCGCAAAAGCACATCGAACTGGGTAGGGTTCAAGTAAAC GGCTCGAAGGTCAAAAACAGTAAAACCATAGACATCGGCGATATTATCGACCTGACGCTC AATTCCCTTCCCTATAAAATCAAGGTTAΆAGGTTTGAACCACCAACGCCGCCCGGCATCC GAGGCGCGGCTTCTGTATGAAGAGGACGCGAAAACGGCAACATTGAGGGAAGAGCGCAAA CAGCTCGACCAATTCAGCCGCATCACTTCCGCCTATCCCGACGGCAGACCGACCAAGCGC GACCGCCGCCAACTGGACAGGCTGAAAAAAGGAGACTGGTAA
NMBl176 Amino acid sequence
MKDKHDSSAMRLDKWLWAARFFKTRSLAQKHIELGRVQVNGSKVKNSKTIDIGDIIDLTL NSLPYKIKVKGLNHQRRPASEARLLYEEDAKTATLREERKQLDQFSRITSAYPDGRPTKR DRRQLDRLKKGDW
NMB1359 Nucleic acid sequence
ATGAACCACACCGTTACCCTGCCCGACCAAACCACCTTTGCCGCCAACGACGGCGAAACC GTTTTGACCGCTGCCGCCCGTCAAAACCTCAACCTGCCCCATTCCTGCAAAAGCGGTGTC
TGCGGACAATGCAAAGCCGAACTGGTCAGCGGCGATATTCAAATGGGCGGACACTCGGAA
CAGGCTTTATCCGAAGCAGAAAAAGCGCAAGGCAAGATTTTGATGTGCTGCACCACTGCG CAAΆGCGΆTATCAACATCAACATCCCCGGCTACAAAGCCGATGCCCTACCCGTCCGCACC CTGCCCGCACGCATCGAAAGTATTATTTTCAAΆCΆCGATGTCGCCCTCCTGAAΆCTTGCC CTGCCCAAAGCCCCGCCGTTTGCCTTCTACGCCGGGCAATACATTGATTTACTGCTGCCG GGCAΆCGTCAGCCGCAGCTACTCCATCGCCAATTTACCCGACCAAGAAGGCATTTTGGAA CTGCACATCCGCAGGCACGAAAACGGTGTCTGCTCGGAAATGATTTTCGGCAGCGAACCC
AAAGTCΆΆAGAAΆAAGGCATCGTCCGCGTTAAAGGCCCGCTCGGTTCGTTTACCTTGCAG GAΆGACAGCGGCAAACCCGTCATCCTGCTGGCΆACCGGCACAGGCTΆCGCCCCCATCCGC AGCATCCTGCTCGACCTTATCCGCCAAGGCAGCAACCGCGCCGTCCATTTCTACTGGGGC GCGCGTCATCAGGATGATTTGTATGCCCTCGAAGAAGCACAAGGGTTGGCATGCCGTCTG AAΆAACGCCTGCTTCACCCCCGTΆTTGTCCCGCCCCGGAGAGGGCTGGCΆGGGΆΆGAAAT GGTCACGTACAAGACATCGCGGCACAΆGACCACCCCGACCTGTCGGAΆTACGΆAGTATTT GCCTGCGGTTCTCCGGCCATGACCGAACAAACAAAGAATCTGTTTGTGCAACAGCATAAG CTGCCGGAAΆΆCTTGTTTTTCTCCGACGCATTCACGCCGTCCGCATCATAA
NMB 1359 Amino acid sequence
MNHTVTLPDQTTFAANDGETVLTAAARQNLNLPHSCKSGVCGQCKAELVSGDIQMGGHSE QALSEAEKAQGKILMCCTTAQSDININIPGYKADALPVRTLPARIESIIFKHDVALLKLA LPKAPPFAFYAGQYIDLLLPGNVSRSYSIANLPDQEGILELHIRRHENGVCSEMIFGSEP KVKEKGIVRVKGPLGSFTLQEDSGKPVILLATGTGYAPIRSILLDLIRQGSNRAVHFYWG ARHQDDLYALEEAQGLACRLKNACFTPVLSRPGEGWQGRNGHVQDIAAQDHPDLSEYEVF ACGSPAMTEQTKNLFVQQHKLPENLFFSDAFTPSAS
NMBl 138 Nucleic acid sequence
ATGAAAGACAAGCACGATTCTTCCGCCATGCGGCTGGACAAATGGCTTTGGGCGGCACGT TTTTTCAAGACCCGTTCCCTTGCGCAAAAGCACATCGAACTGGGTAGGGTTCAAGTAAAC GGCTCGAAGGTCAAAΆΆCAGTAAAACCATAGACATCGGCGATATTATCGACCTGACGCTC AATTCCCTTCCCTATAAAATCAAGGTTAAAGGTTTGAACCΆCCAACGCCGCCCGGCATCC GAGGCGCGGCTTCTGTATGAAGAGGACGCGAAAACGGCAACATTGAGGGAAGAGCGCAAA CAGCTCGACCAATTCAGCCGCATCACTTCCGCCTATCCCGACGGCAGACCGACCAAGCGC GACCGCCGCCAACTGGACAGGCTGAAAΆΆAGGAGACTGGTAA
NMBl138 Amino acid sequence
MKDKHDSSAMRLDKWLWAARFFKTRSLAQKHIELGRVQVNGSKVKNSKTIDIGDIIDLTL NSLPYKIKVKGLNHQRRPASEARLLYEEDAKTATLREERKQLDQFSRITSAYPDGRPTKR DRRQLDRLKKGDW
Schedule of SEQ ID Nos
SEQ BD No Sequence
1 NMB0341 DNA
2 NMB0341 Protein
3 NMB 1583 DNA
4 NMB 1583 Protein
5 NMB1345 DNA
6 NMB 1345 Protein 7 NMB0738 DNA
8 NMB 0738 Protein
9 NMB0792 DNA
10 NMB0792 Protein
11 NMB0279 DNA
12 NMB0279 Protein
13 NMB2050 DNA
14 NMB2050 Protein
15 NMB1335 DNA
16 NMB 1335 Protein
17 NMB2035 DNA
18 NMB2035 Protein
19 NMB1351 DNA
20 NMB 1351 Protein
21 NMB 1574 DNA
22 NMB 1574 Protein
23 NMB 1298 DNA
24 NMB 1298 Protein
25 NMB 1856 DNA
26 NMB 1856 Protein
27 NMBOl 19 DNA
28 NMBOl 19 Protein
29 NMB 1705 DNA
30 NMB 1705 Protein
31 NMB2065 DNA
32 NMB2065 Protein
33 NMB0339 DNA
34 NMB 0339 Protein
35 NMB0401 DNA
36 NMB0401 Protein
37 NMB 1467 DNA
38 NMB 1467 Protein 39 NMB2056 DNA
40 NMB2056 Protein
41 NMB0808 DNA
42 NMB0808 Protein
43 NMB0774 DNA
44 NMB0774 Protein
45 NMA0078 DNA
46 NMA0078 Protein
47 NMBO337 DNA
48 NMB0337 Protein
49 NMB0191 DNA
50 NMB0191 Protein
51 NMB 171 O DNA
52 NMB 1710 Protein
53 NMB0062 DNA
54 NMB0062 Protein
55 NMB1333 DNA .
56 NMB1333 Protein
57 NMB0377 DNA
58 NMB0377 Protein
59 NMB0264 DNA
60 NMB0264 Protein
61 NMB 1036 DNA
62 NMB 1036 Protein
63 NMB 1176 DNA
64 NMB 1176 Protein
65 NMB 1359 DNA
66 NMB 1359 Protein
61 NMBl 138 DNA
68 NMB 1138 Protein

Claims

1. A polypeptide comprising the amino acid sequence selected from any one of SEQ ID Nos 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68;
or a fragment or variant thereof or a fusion of such a fragment or variant.
2. A polynucleotide encoding a polypeptide according to Claim 1.
3. A polypeptide according to Claim 1 or polynucleotide according to Claim 2 for use in medicine.
4. A polypeptide according to Claim 1 or polynucleotide according to Claim 2 for use in a vaccine.
5. A method for making a polypeptide according to Claim I5 the method comprising expressing the polynucleotide of Claim 2 in a host cell and isolating said polypeptide.
6. A method for making a polypeptide according to Claim 1 comprising chemically synthesising said polypeptide.
7.. A method of vaccinating an individual against Neisseria meningitidis, the method comprising administering to the individual a polypeptide according to Claim 1 or a polynucleotide according to Claim 2. ■
8. Use of a polypeptide according to Claim 1 or a polynucleotide according to Claim 2 in the manufacture of a vaccine for vaccinating an individual against Neisseria meningitidis.
9. A pharmaceutical composition comprising a polypeptide according to Claim 1 or a polynucleotide according to Claim 2 and a pharmaceutically acceptable carrier.
EP05823115A 2004-12-23 2005-12-23 Vaccines against neisseria meningitidis Withdrawn EP1848457A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
PCT/GB2004/005441 WO2005060995A2 (en) 2003-12-23 2004-12-23 Identification of antigenically important neisseria antigens by screening insertional mutant libraries with antiserum
PCT/GB2005/005113 WO2006067518A2 (en) 2004-12-23 2005-12-23 Vaccines against neisseria meningitidis

Publications (1)

Publication Number Publication Date
EP1848457A2 true EP1848457A2 (en) 2007-10-31

Family

ID=36282716

Family Applications (1)

Application Number Title Priority Date Filing Date
EP05823115A Withdrawn EP1848457A2 (en) 2004-12-23 2005-12-23 Vaccines against neisseria meningitidis

Country Status (10)

Country Link
EP (1) EP1848457A2 (en)
JP (1) JP2008525008A (en)
KR (1) KR20070094762A (en)
CN (2) CN101115502A (en)
AU (1) AU2005317835A1 (en)
CA (1) CA2592156A1 (en)
MX (1) MX2007007886A (en)
NO (1) NO20073256L (en)
RU (1) RU2007127921A (en)
WO (1) WO2006067518A2 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090226479A1 (en) * 2005-12-23 2009-09-10 Imperial Innovations Limited Vaccines and their use

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9814902D0 (en) * 1998-07-10 1998-09-09 Univ Nottingham Screening of neisserial vaccine candidates against pathogenic neisseria

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2006067518A2 *

Also Published As

Publication number Publication date
RU2007127921A (en) 2009-01-27
CN101370514A (en) 2009-02-18
JP2008525008A (en) 2008-07-17
CN101115502A (en) 2008-01-30
MX2007007886A (en) 2008-01-16
AU2005317835A1 (en) 2006-06-29
WO2006067518A3 (en) 2006-11-23
CA2592156A1 (en) 2006-06-29
NO20073256L (en) 2007-09-17
WO2006067518A2 (en) 2006-06-29
KR20070094762A (en) 2007-09-21

Similar Documents

Publication Publication Date Title
AU2009329782B2 (en) Modified Streptococcus pneumonia pneumolysin (PLY) polypeptides
JPH10513349A (en) Microbial protein, microorganism producing the protein, and use of the protein in vaccine and tuberculosis detection
PL197449B1 (en) Basb029 polynucleotide(s) and polypeptides from neisseria meningitidis
NZ532297A (en) Virulence genes, proteins, and their use in treating conditions associated with infection by Neisseria or gram-negative bacteria
CN107349423B (en) Meningococcal antigen combination and application thereof
US6835384B1 (en) Nucleic acids and polypeptides specific of the neisseria genus pathogenic strains
WO2006067518A2 (en) Vaccines against neisseria meningitidis
US20080138357A1 (en) Vaccines Against Neisseria Meningitidis
US20090226479A1 (en) Vaccines and their use
US20070275017A1 (en) Identification of Antigenically Important Neisseria Antigens by Screening Insertional Mutant Libraries with Antiserum
WO2009094006A2 (en) Bordetella outer-membrane protein antigens and methods of making and using the same
US20030049265A1 (en) Heliobacter pylori antigen
JP2004534524A (en) Pathogenic and symbiotic vaccine antigens
CA2547537A1 (en) Protein nmb0928 and use thereof in pharmaceutical formulations
US20090208521A1 (en) Pharmaceutical compositions containing protein nma0939
RU2336900C2 (en) Nmb1125 protein and its application in pharmaceutical compositions
CA2647465A1 (en) Pharmaceutical composition containing the nmb0606 protein
EP1980266A1 (en) New virulence factors of Streptococcus pneumoniae
CA2241023A1 (en) Novel polynucleotides and polypeptides in pathogenic mycobacteria and their use as diagnostics, vaccines and targets for chemotherapy
NZ502423A (en) Polynucleotide sequences, designated GS, in pathogenic mycobacteria and their use in vaccines

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI SK TR

17P Request for examination filed

Effective date: 20070719

REG Reference to a national code

Ref country code: HK

Ref legal event code: DE

Ref document number: 1106707

Country of ref document: HK

DAX Request for extension of the european patent (deleted)
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

18W Application withdrawn

Effective date: 20080126

REG Reference to a national code

Ref country code: HK

Ref legal event code: WD

Ref document number: 1106707

Country of ref document: HK