MX2007007886A - Vaccines and their use. - Google Patents

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 <i>Neisseira meningitidis.</i>.

Description

VACCINES AGAINST NEISSERIA EN1NG1TIDIS DESCRIPTIVE MEMORY The present invention relates to vaccines and their use, and in particular to vaccines for meningococcal disease. The listing or discussion of a document previously published in this specification should not necessarily be taken as a knowledge that the document is part of the state of the art or is common general knowledge. The documents listed in the specification are incorporated in the present invention as references. 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 antimicrobial agents such as antibiotics. Vaccination provides an alternative method to combat microbial infections, but it is often difficult to identify such immunogens for use in vaccines which are safe and effective against a range of different isolates of a pathogenic microorganism, particularly a genetically diverse microorganism. Although it is possible to develop vaccines which use as the immunogen substantially intact microorganisms, such as live attenuated bacteria that typically contain a mutation or mutations in a gene virulence determinant, not all microorganisms are suitable for carrying out this method, and it is not always desirable to adopt this method for a particular microorganism where safety can not always be guaranteed. Also, some microorganisms express molecules that limit host proteins, and these are undesirable in a vaccine. A particular group of microorganisms for which it is important to develop additional vaccines is Neisseria meningitidis which causes meningococcal disease, a life-threatening infection which in Europe, North America, developed countries and in some other places remains an important cause of mortality despite the introduction of serogroup C conjugated polysaccharide vaccine. This is because infections caused by strains of serogroup B (NmB), which express a capsule of polysialic acid associated with a-8, are still frequent. The term "serogroup" in relation to N. meningitidis refers to the polysaccharide capsule that is expressed in the bacterium. The common serogroup in the UK that causes the disease is B, while in Africa it is A. Meningococcal septicaemia who continues to produce a high proportion of fatal cases; and survivors are often left with severe psychological and / or physical disabilities. After an unspecified prodromal disease, meningococcal septicemia can present as a fulminating disease that is refractory to appropriate antimicrobial therapy and measures for total life support. Therefore, the best method to combat the threat to public health of meningococcal disease is through prophylactic vaccination. Early nonspecific clinical signs and the fulminating course of meningococcal infection mean that therapy is often inefficient. Therefore, vaccination is considered the most effective strategy to reduce the weight of the global disease caused by this pathogen (Feavers (2000) ABC of meningococcal diversity, Nature 404, 451-2). The existing vaccines to prevent infections by N. meningitidis serogroup A, C, W135, and Y are based on the polysaccharide capsule located on the surface of the bacteria (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 Infecí Dis. , 200-4; Lieberman et al (1996). Safety and immunogenicity of a serogroups A / C Neisseria meningitis oligosaccharide-protein conjugate vaccine in young children. A randomized controlled trial. J. American Med. Assoc. 275, 1499-1503). Progress towards a vaccine against serogroup B infections has been more difficult since its capsule, a sialic acid homopolymer associated with oc2-8, is a relatively poor immunogen in humans. This is because it shares epitopes expressed in. a human cell adhesion molecule, N-CAM1 (Finne et al (1983) Antigenic similarities between brain components and bacteria causing meningitis. Implications for vaccine development and pathogenesis. Lancet 2, 355-357). In fact, the generation of immune responses against the serogroup B capsule could actually provide damage. Therefore, a need remains for new vaccines to prevent N. meningitidis infections of serogroup B. The most validated immunological protection correlation against meningococcal disease is the serum bactericidal assay (SBA). The SBA evaluates the ability of antibodies (usually subglass lgG2a) in serum to mediate the complete arrangement on the bacterial cell surface, assembly of the complex to attack the membrane, and bacterial lysis. In SBA, a known number of bacteria are exposed to serial dilutions of sera with a defined source of complement. The number of surviving bacteria is determined, and the SBA is defined as the reciprocal of the highest dilution of serum that mediates 50% elimination. The SBA predicts protection against serogroup C infections, and has a very wide use as a substitute for immunity against NmB infections. Importantly, SBA is an available marker of immunity for pre-clinical evaluation of vaccines, and provides an adequate endpoint in clinical trials. Most efforts to develop a vaccine for NmB are directed towards the definition of effective protein subunits. There has been a strong investment in "reverse vaccination", in which genomic sequences are interrogated for proteins that are potentially expressed on the surface which are expressed as heterologous antigens and evaluated for their ability to generate important responses in animals. However, this method is limited by 1) computer algorithms to predict antigens that are expressed on the surface, 2) inability to express many of the potential immunogens, and 3) total safety in murine immune responses. The key to a successful vaccine is to define the antigen (s) that induce protection against a wide range of disease isolates regardless of the serogroup or clonal group. A method of genetic selection (which has been determined by the inventors as Genetic Screening for Immunogens or GSI) was used to isolate antigens that are conserved through the genetic diversity of strains. microbials and this is exemplified in relation to the meningococcal strains. This was done by identifying microbial antigens, such as the N. meningitidis antigens, by GSI as described in more detail below; and it was validated by evaluating the function of the immune response induced by the recombinant antigens and by evaluating the protective efficiency of the antigens (see the examples and see PCT / GB2004 / 005441 (published as WO 2005/060995). July 7, 2005) incorporated in the present invention as reference). In essence, the GSI method refers to a method for identifying a polypeptide of a microorganism said 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 mutants other than the microorganism; (2) contacting the plurality of mutant microorganisms with antibodies from an animal which have generated an immune response to the microorganism or a part thereof, under conditions where if the antibodies bind to the mutant microorganism the mutant microorganism is eliminated; (3) selecting surviving mutant microorganisms from step (2); (4) identify the gene that contains the mutation in any surviving mutant microorganism; and (5) identifying the polypeptide encoded by the gene. It would appear that by the way in which polypeptides are identified, these are highly relevant as antigenic polypeptides. As described in more detail in the examples, the particular genes identified by the GSI method are the genes NBM0341 (TspA), NMB0338, NMB1345, NMB0738, NBM0792 (NadC family), NMB0279, NMB2050, NMB1335 (CreA), NMB2035, NMB1351 (Fmu and Fmv), NMB1574 (llvC), NMB1298 (rsuA), NMB1856 (LysR family), NMB0119, 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 , NMBI 176, NMB1359 and NMB1 138 from Neisseria meningitidis. The genomic sequence for N. meningitidis is available, for example from The Institute of Genome Research (TIGR); www.tigr.org Although these genes are part of the genome that has been sequenced, as far as the knowledge of the inventors, 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. Therefore, the invention includes the isolated genes as mentioned above and in the examples and variants and fragments and fusions of said variants and fragments, and includes the polypeptides that the genes encode as described above, together with variants and fragments of the themselves, and mergers of said fragments and variants. The variants, fragments and mergers are described in more detail below. Preferably, the variants, fragments and fusions of the genes given above are those that encode a polypeptide which gives rise to neutralizing antibodies against N. meningitidis. Similarly, preferably, the variants, fragments and fusions of the polypeptide whose sequence was given above are those that give rise to neutralizing antibodies against N. meningitidis. Neutralizing antibodies can 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 provide in the example (preferably the isolated coding region) or that code the brave, fragments or fusions. The invention also includes expression vectors comprising said polynucleotides and host cells comprising said polynucleotides and vectors (as described in greater detail below). The polypeptides described in the examples are antigens identified by the method of the invention. Molecular biology 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 can be made, for example by identifying the related genes in other microorganisms or in other strains of the microorganism, and by cloning, isolating or synthesizing the gene. Typically, gene variants are those that have at least 70% sequence identity, more preferably at least 85% sequence identity, more preferably at least 95% sequence identity with the genes as provided above. Of course, replacements, deletions and insertions can be tolerated. The degree of similarity between one and the other nucleic acid sequence can be determined using the GAP program of the University of Wisconsin Computer Group. The variants of the gene are also those that hybridize under severe conditions with the gene. By "severe" the inventors refer to the hybrid gene with the probe when the gene is immobilized on a membrane and the probe (which, in this case is> 200 nucleotides in length) is in solution and the immobilized gene / hybridized probe is washed in 0.1 x SSC at 65 ° C for 10 minutes. SSC is NaCl 0.15 M / 0.015 M Na citrate. Gene fragments (or variant gene) can be made which are, for example, 20% or 30% or 40% or 50% or 60% or 70% or 80% or 90% of the total gene. Preferred fragments include all or a part of the coding sequence. The variant and fragments can be fused with other unrelated polynucleotides. The polynucleotide encodes a polypeptide which is immunogenic and reactive with the antibodies from an animal which has been subjected to the microorganisms from which the gene has been identified. The antigen can be the polypeptide as it is encoded by the gene previously identified, and the polypeptide sequence can be easily deduced from the gene sequence. In the further embodiments, the antigen may be a fragment of the identified polypeptide or it may be a variant of the identified polypeptide or it may be a fusion of the polypeptide or fragment or variant. Therefore, a particular aspect of the invention provides a polypeptide comprising the amino acid sequence selected from any 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 said fragment or variant. Therefore, the invention provides the following isolated proteins, or fragments or variants thereof, or fusion thereof: NMB0341, NMB1583, NMB1345, NMB0738, NMB0792, NMB0279, NMB2050, NMB1335, NMB2035, NMB1351, NMB1574, NMB1298, NMB1856 , NMB01 19, NMB1705, NMB2065, NMB0339, NMB0401, NMB1467, NMB2056, NMB0808, NMB0774, NMA0078, NMB0337, NMB0191, NMB1710, NMB132, NMB1333, NMB0377, NMB0264, NMB1036, NMB1176, NMB1359 and NMB1 138 as described below. The fragments of the identified polypeptide can be made which are, for example, 20% or 30% or 40% or 50% or 60% or 70% or 80% or 90% of the total polypeptide. Typically, the 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 can be made. By "variants" the inventors include insertions, deletions and substitutions, either conservative or non-conservative, wherein said changes do not substantially alter the normal function of the protein. By "conservative substitutions" are meant combinations such as Gly, Ala; Val, He, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. Such variant can be made using the well-known methods of protein design and site-directed mutagenesis. A particular class of variants are those encoded by variable 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, more preferably at least 95% sequence identity with the polypeptide identified using the method of the invention. The percentage sequence identity between two polypeptides can 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 the percentage identity is calculated in relation to the polypeptides whose sequence has been aligned optimal way. Alignment can be carried out alternatively using the Clustal W program (Thompson et al, (1994) Nucleic Acids Res 22, 4673-80). The parameters used can be as follows: Parameters of fast par-to-par alignment: size K-tuple (word); 1, window size; 5, penalty for space; 3, number of diagonals in the upper part; 5. Evaluation method: x percent. Parameters of multiple alignment: sanction for open space; 10, penalty for extension of space; 0.05. Evaluation matrix: BLOSUM. The fusions can be fusions with any suitable polypeptide. Typically, the polypeptide is one which is capable of improving the immune response to the polypeptide to which it is fusing. The fusion partner can a polypeptide that facilitates purification, for example by forming a binding site for a portion that can be immobilized in, for example, an affinity chromatography column. Therefore, the fusion partner may comprise oligo-histidine or other amino acids which bind cobalt or nickel ions. This may also be an epitope for a monoclonal antibody such as a Myc epitope. As discussed above, polypeptide or polypeptide fragment variants, or fusions thereof, are typically those that give rise to neutralizing antibodies against N. meningitidis. Therefore, the invention also includes, a method for making an antigen as described above, and antigens that are obtained or obtained by the method. The polynucleotides of the invention can be cloned into vectors, such as expression vectors, as is well known in the art. Such vectors can be present in host cells, such as bacterial cells, yeast, mammalian cells and insect cells. The antigens of the invention can be easily expressed from the polynucleotides in a suitable host cell, and can be isolated from them for use in a vaccine. Typical expression systems include the commercially available pET expression vector series and the E. coli host cells such as BL21. The expressed polypeptides can be purified by any method known in the art. Conveniently, the antigen is operated on a fusion partner that binds to an affinity column as discussed above, and the function is purified using the column of affinity (for example 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 suitable for use as a vaccine. In that case, the antigen is purified from the host cell in which it is produced (or if it is produced by peptide synthesis, it is purified from any contaminants in the synthesis). Typically the antigen contains less than 5% contaminating material, preferably less than 2%, 1%, 0.5%, 0.1%, 0.01%, before it is formulated for use in a vaccine. The desirable antigen is substantially free of pyrogens. Therefore, the invention additionally includes a vaccine comprising the antigen, and the method for making a vaccine comprising combining the antigen with a suitable vehicle, such as a saline solution with pH regulated with phosphate. Although 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 vehicle (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 vehicles will be water or saline solution which will be sterile and free of pyrogens. The vaccine may also conveniently include an adjuvant. Active immunization of the patient is preferred. In this method, one or more antigens are prepared in an immunogenic formulation that it contains suitable adjuvants and vehicles and is administered to the patient in known ways. Suitable adjuvants include complete or incomplete Freund's adjuvant, muramyl dipeptide, the "Iscoms" of EP 109 942, EP 180 564 and EP 231 039, aluminum hydroxide, saponin, DEAE-dextran, neutral oils (such as miglyol), oils plants (such as arachis oil), liposomes, Pluronic polyols or the Ribi adjuvant system (see, for example GB-A-2 189 141). "Plurónico" is a registered trademark. The patient to be immunized is a patient that needs 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 thereof, or a polynucleotide of the invention or a variant or fragment thereof or a fusion thereof, and a pharmaceutically acceptable carrier as It was discussed early. The aforementioned antigens of the invention (or polynucleotides encoding said antigens) or a formulation thereof can be administered by any conventional method including oral and parenteral injection (e.g., subcutaneous or intramuscular). The treatment may consist of a particular dose or of a plurality of doses over a period of time. It will be appreciated that the vaccine of the invention, depending on its antigen (or polynucleotide) component, 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, be 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. Therefore, 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 can be used as the sole antigen in a vaccine or can be used in combination with other antigens either directed to the same microorganisms or to different microorganisms of the disease. In relation to N. meningitidis, the antigen obtained which is reactive against NmB can be combined with components used in vaccines for serogroups A and / or C. This can also be conveniently combined with antigenic components that provide protection against Haemophilus and / or Streptococcus pneumoniae The additional antigenic components may be polypeptides or may be other antigenic components such as a polysaccharide. Polysaccharides can 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 aforementioned vaccines and vaccination methods 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) which encodes said antigen), and that the disease to be vaccinated against is the infection by Neisseria meningitidis (meningococcal disease). The invention will now be described in greater detail with reference to the following non-limiting examples.

EXAMPLE 1 Genetic selection for immunogens (GSI) in N. meningitidis The application of GSI in this example includes libraries of selection of insertion mutants of N. meningitidis for strains that are less susceptible to elimination by bactericidal antibodies. GSI is deciphered in more detail in PCT / GB2005 / 005441 (published as WO 2005/060995 on July 7, 2005). The inventors have demonstrated the effectiveness of GSI in selecting a library of mutants of the sequenced NmB isolate, MC58, with the sera generated in mice against one less capsule of the same strain. A total of 40,000 mutants were analyzed with serum generated in mice by intraperitoneal immunization with the homologous strain; the SBA of this serum is around 2,000 against the wild-type strain. The surviving mutants were detected when the library was exposed to the serum at a 1: 560 dilution (which eliminates all wild-type bacteria). To establish whether the insertion of the transposon in the surviving mutants was responsible for the ability to resist elimination, the mutations were backcrossed into the parental strain, and the backcrossed mutants were confirmed as more resistant to elimination 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. The inventors found that two of the affected genes were TspA and NMB0338. TspA is a surface antigen which induces strong CD4 + T cell responses and is recognized by serum 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 on the cell surface. The amino acid sequence encoded by NMB0338 is: MERNGVFGKIVGi i RMSSEHAaASYPKPCKSF RQSWFRVRSC GGVFIYGA MMKLIYTVIKIII LLFL IAVINTDAVTFSYLPGQ FDLP IVVLFGAGVVGI? FGMFAI.FGRDLSLRGBNGR WyeVKKNAKLTGKELTAPPAQNAPESTKQP There are several practical advantages to the use of NmB for GSI in addition to the public health imperative: a) the bacteria can be treated in a manageable genetic manner; b) the elimination of the bacteria by an immune effector mechanism is direct to the assay; c) genomic sequences are available for three isolates from different serogroups and clonal lineages (IV-A, ET-5, and ET-37 for serogroups A, B5 and C, respectively); and d) well-characterized clinical resources available for this work. GSI has two potential limitations. First, targets for bactericidal antibodies can be essential. This is unlikely since all known targets of bactericidal antibodies in NmB are not essential, and the currently unlicensed bacterial vaccine targets an essential gene product. Second, the sera will contain antibodies to multiple antigens, and, the loss of a particular antigen may not affect the survival of the mutants. The inventors have already shown that even during the selection with the serum generated against the homologous strain, the relevant antigens had already been identified using appropriate dilutions of the serum. The main advantages of GSI are that 1) the high resolution steps do not include technically demanding or expensive procedures (such as protein expression purification and immunization), and 2) human samples can be used in the assay instead of just relying on the animal data. GSI will quickly determine precisely the subpopulation of surface proteins that induce bactericidal activity, allowing more detailed analysis of a smaller number of candidates. 1. Identification of bactericidal antibody targets using GSI Murine sera generated against heterologous strains, and human sera, are used to identify cross-reactive antigens. The sera are obtained from: i) mice immunized 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) sera from patients with acute and convalescent disease infected with known isolates of N. meningitidis (provided by Dr R. Wall, Northwick Park) ii) pre- and post-immunization samples (provided by the Meningococcal Reference Laboratory) from volunteers who received vaccines with defined outer membrane vesicles (OMVs) derived from the NmB isolate, H44 / 76. Each of these sources of sera has specific advantages and disadvantages. a) Serums from animals immunized with heterologous strains (for example the sequenced strains of serogroup A or C) are used in GSI to select the MC58 mutant library. The inventors have shown that immunization with live, attenuated NmB induces responses to the bactericidal antibody with cross-reactivity against the serogroup A and C strains. The antigen absent in mutants with improved survival with respect to human serum is identified by rescue of the altered gene marker. b) Mutations are identified that confer resistance against elimination by the heterologous serum, and it is determined whether the gene product is also a target for the elimination of the sequenced strains, serogroup A and C, Z2491 and FAM18 respectively. The genome databases are inspected for gene homologs. If a homologue is present, the transposon insert was amplified from the MC58 mutant and introduced into the strains of serogroup A and C by transformation. The relative survivals of the mutant strain and the wild-type strain of each serogroup were compared. Therefore, GSI can provide information quickly if the targets of bactericidal activity are conserved and accessible in various strains of N. meningitidis, regardless of the serogroup immunotype and subserotype. c) mutants with improved survival against the sera generated in mice are evaluated using human serum from either convalescent or vaccinated patients who received the heterologous OMV vaccines (derived from H44 / 76). This addresses the important question of whether whites are capable of inducing bactericidal antibodies in humans. With other methods of vaccination, this information is only obtained in the later stage, costly clinical trials that require the development of candidates for GMP vaccine. The advantages are that GSI is a high resolution analysis that is carried out using simple, available techniques. Antigens that induce bactericidal antibodies in humans and that mediate the elimination of Multiple strains can be quickly identified as GSI if they are flexible with respect to the bacterial strains and sera used. The mutants selected using human serum were analyzed in the same manner as those selected by murine serum. 2. Evaluation of the antibody response of recombinant antigens to GSI Proteins that are targets of bactericidal antibodies that are recognized by serum 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 the expression of the protein under the control of a T7 promoter or produced by arabinose., respectively. The purification of the recombinant proteins from the total cellular protein is carried out via the His tag fused to the C terminus of the protein on a nickel or cobalt column. Adult New Zealand white mice were immunized on two separate occasions for four weeks by subcutaneous injection with 25 ug of purified protein with incomplete Freund's adjuvant. Sera from animals will be evaluated prior to immunization for pre-existing anti-Mn antibodies by whole-cell ELISA. Animals that have an initial serum titre of <; 1: 2 were used to Immunization experiments. The post-immunization sera were obtained two weeks after the second immunization. To confirm that the specific antibodies had been generated, the pre- and post-immunization sera were evaluated by i) Western analysis against the purified protein and ii) ELISA using cells from the wild-type organism and the corresponding mutant (generated by GSI ). SBAs will be carried out against MC58 (the homologous strain), and serogroup A and C strains sequenced with the rabbit immune serum. The test will be carried out in triplicate on at least two occasions. The SBAs of > 8 will be considered significant. The results provide evidence as to whether protein candidates can induce bactericidal antibodies as recombinant proteins. 3. Establishing the Protective Efficiency of GSI Antigens All candidates were evaluated for their ability to protect animals against the test with live bacteria since this allows any aspect of immunity (cellular or humoral) to be absent in the particular assay . The inventors have established a model of active immunization and protection against infection by living bacteria. In this model, the adult mice were immunized on days 0 and 21, and on day 28 they received a test with live bacteria of 106 or 107 CFU of MC58 intraperitoneally on iron dextran (as the iron source supplement). The model is similar to that described for the evaluation of Protective efficiency of immunization with Tbps Danve et al (1993) Vaccine 11, 1214-1220. Unimmunized animals developed bacteremia within the first 4 hours of infection, and showed signs of systemic disease at 24 hours. The inventors have already been able to demonstrate the protective efficiency of both attenuated Nm strains and a proteinaceous antigen against the live meningococcal test; PorA is an outer membrane protein that induces bactericidal antibodies, but which is not a leading vaccine candidate due to the extensive antigenic variation (Bart et al (1999) Infecí Immun 67, 3832-3846). Six-week-old BALB / c mice (group size, 35 animals) received 25 ug of recombinant protein with incomplete Freund's adjuvant subcutaneously at days 0 and 21, then tested with 106 (15 animals) or 107 (15 animals) CFU of MC58 intraperitoneally at day 28. Two test doses were used to examine the efficiency of the vaccine at a high and low test dose; the sera were obtained at day 28 from the five animals remaining in each group, and from five animals before the first immunization and stored at -70 ° C for additional immunological assays. The animals in the control groups received either i) adjuvant alone, ii) refolded recombinant PorA, and iii) a live, attenuated strain of Nm. To reduce the general number of animals in the control groups, a series of five candidates will be evaluated at the same time (number of groups = 5 candidates + 3 controls). The survival of the animals in the groups was compared using the Mann Whitney U test.

With group sizes of 15 mice / dose, the experiments are trained to show a 25% difference in survival between the groups. For vaccines that show significant protection against the test, a repeated experiment is carried out to confirm the finding. In addition, to establish that vaccination with a candidate also induces protection against bacteremia, the levels of bacteremia are determined during the second experiment; Blood samples are taken for 22 hours post-infection in immunized and non-immunized animals (bacteremia is maximum at this time). The results are performed using a two-tailed Student's T test to determine if there is a significant reduction in bacteremia in vaccinated animals.

Additional materials and methods used Mutagenesis of Neisseria meningitidis For work with Neisseria meningitidis, the mutants were constructed by in vitro mutagenesis. Genomic DNA from N. meningitidis was mutagenized with a Tn5 derivative containing a marker that encodes kanamycin resistance, and an origin of replication which is functional in E. coli. These elements are joined by the ends composed of Tn5. The transposition reactions were carried out with an overactive variant of Tn5 and the DNA was repaired with T4 DNA polymerase and ligase in the presence of ATP and nucleotides. The repaired DNA was used to transform N. meningitidis into an organism resistant to kanamycin The Southern analysis confirmed that each mutant contained a particular insertion of the transposon only.

Serum bactericidal assays (SBAs) Bacteria were grown overnight in solid medium (Brain heart infusion medium with Levanthals supplement) and then re-seeded in solid medium for four hours the morning of the experiments. After this time, the bacteria were harvested in saline with pH regulated with phosphate and numbered. SBAs were carried out in a volume of 1 ml, which contained a complement source (rabbit or human) and approximately 105 colony forming units. The bacteria were harvested at the end of the incubation and plated on solid medium to recover the surviving bacteria.

Isolation of transposon insertion sites Genomic DNA will be recovered from mutants of interest by standard methods and will be digested with Pvull, EcoRV, and Dral for three hours, then purified by phenol extraction. Then, the DNA will be self-ligated in a volume of 100 microliters overnight at 16 ° C in the presence of T4 DNA ligase, precipitated, then used to transform E. coli towards organisms resistant to kanamycin by electroporation.

EXAMPLE 2 Additional evaluation and results thereof GSI has been used to select a library of approximately 40,000 MC58 insertion mutants. The library was constructed by in vitro mutagenesis of Tn5, using a transposon containing the origin of replication from pACYC184. MC58 was chosen since it is an isolated sero group B from N. meningitidis, and the complete genomic sequence of this strain is known. The library is always selected in parallel with the wild-type strain as a control, and the number of colonies recovered from the library and the wild-type colonies is shown.

Selection with murine sera The library was initially analyzed using serum from animals immunized with the attenuated YH102 strain. Adult mice (Balb / C) received 108 colony forming units intraperitoneally on three occasions, and sera were collected 10 days after the final immunization. The selection identified several mutants with improved resistance to elimination by serum: This was confirmed by isolation of individual mutants, reconstruction of the mutation in the original genetic background, and reassessment of the individual mutants by their susceptibility analysis mediated by the complement against the wild-type organisms. The transposon inserts are in the following gene: MB0341 (? SpA) DNA sequence ATGCCCCCCGGCCGAC? GCCCCGCCG? TGCCCGATGATGRCGAAATTTACAGACTGTACG CGGTCAA ^ GCC? Ar7CAGCCGCC.AACCCACAGGGGAACATCTTG. ^. ^ AAaCAa.CAGACA ATCAAACTGA7TGCCGCCTCCGTCGCAGTTGCCGCATCCTTTCAGGCACATGCTGGACTG GGCGGACTGAA A CCAGTCCAACCT? G? CGAACCCTTTTCCGGCAGCATTACCGTAACC GGCGAftGAAGCCAAAGCCCTGCTAGGCGGCGGCAGCGTTACCGTTTCCGAAAAAGGCCTG ACCGCCAAACTCCACAAGTTGGGCGACAftAGCCGTCATTGCCGTTTCT CCGA? CAGGCA GTCCGCGATCCCGTCCTGGTGrTCCGCATCGGCGCAGGCGCACAGGTACGCGAATACACC GC ^ TCCTCGATCCTGTCGGCTAC CGCCCAAAACCW? TCTGCACTTTCAGACdGCAAG ACACACCGaVü? CCGCTCCGACAGCAGAGTCCCA? G / ^ J ^ lAAATCAAAACGCCAAAGCCCTC CGCAAAACCGATAJs GACAGCGCGACGCAGCCG? CAAACCGGCATACA.CGGCiAA ACCCATACCGTCCGCAñAGGCGAAACGGTC? IAACAGATTGCCßCCGCCATCCGCCCGAAA CACCTGACGCTCGñACAGGTTGCCGATGCGCTGCTGAAGGCAAACCCAAñTGTTTCCGCA CACGGCAGACTGCGTGCGGGCAGCGTGCTTCACATTCCGAATCTGAACAGGATCAAAGCG GAACAACCCAAACCGO ^ ACGGCGAAACCCAAAGCCGRAACCGCATCCATGCCGTCCGAA CCGTCCAA? CAGGCAftCGGTAGAGAAACCGGTTGAAaAACCTGAAGCAAAAGTTGCCGCG CCCGAftGCAAAAGCGGA »AAACCGGCCGTTCGACCCGAACCTGTACCCGCTGCAAATACT GCCGCATCGG? AACCGCTGCCGAATCCGCCCCCCAAGAAGCCGCCGCTTCTGCCATCGAC ACGCCGACCGACGAAacCGGTAACGCCGTTTCCGAACCTGTCGAACAGGTTTC? GCCGAA GAAGAAACCGAAi \ GCGGACTGTTTGACGGTCTGTTCGGCGGTTCG? ACACCTTGCrGCTT GCCGGCGGAGGCGCGGCATTAATCGCCCTGCTGCTGCTTTTGCGCCTTGCCCAA CCAAA CGCGCGCGCCGTACCGAAGAATCCGTCCCTGAGGAAGAGCCTGACCTTGACGACGCGGCA G ^ CGACGGCATñGAAñTCACCTTTGCCGAAGTCGAftACTCCGGCACGCCCGAACCCGCZ CCGA? AAACGATGTAAACGACACACTTGCC tAGATGGGGAATCTGAAGAAGAGTTATCG GCA? AACAAACG? TCGAGTCGAAACCGATACGCC? TCCAACCGCATCGACTTGGATTTC GACAGCCTGGCAGCC GCGCAAAACGGCATTTTATCCGGCGCACTTACGCAGGATGAAGAA? CCCAAAAACGCGCGGATGCCGATTGGAACGCC? TCGATCCACAGACAGCG GTACGAG CCCGAGACCTTCAACCCG7ACAACCCTGTCGAfiATCGTCATCGACa.CGCCCGAACCGGftA TCTGTCGCCC? AACTGCCGAAAACAAACCGGA'iñCCGTCGATACCGATTTCTCCG? CAAC CTGCCCTCAAACA? CCATATCGGCACAGAAGAA? CAGCTTCCGCAAAACCTGCCTCACCC TCCGGACTGGCAGGC7TCCTGAAGGCTTCCTCGCCCGAAACCATCTTGGAAAAAACAGTT GCCGAAGTCCAftACACCGGAAGAGTTGCACGATTTCCTGAAAGTGTACGAAACCGATGCC GTCGCGG ?? ACTGCGCCTGAAACGCCCGAT? TCAACGCCGCCGCAGACGATTTGTCCGCA TTGCTTCAACCTGCCGAAGCACCGTCCGTTGAGGA? LñATATAACGGAAACCGTTGCCGAA ACACCCGACTTCAACGCCACCGCAGACGATT GTCCGCATTACTTCAACCTTCTAAAGTA CCTGCCGTTGAGGAAAATGCAGCGGAAACCGTTGCCGATGATTTGTCCGCACTGTTGCAA CCTGCTGAAGCACCGGCCGTTGAGGAAAñTGTAACGGAASCCGTTGCCGAACACCCGAT TTCAACGCCACCGCAGACGATTTGTCCGCATTACTTCAACCTTCTGAAGCACCTGCCGT? GAGGAAAATGCAGCGG? AACCGTTGCCGATGATtTGTCCGCACTGTTGCAACC GCTGWi GCACCGGCCGTTGAGGAAAATGCAGCGGAAATCACTTTGGAAACGCCTGATTCCAACACC TCTGA6GCAGACGCTTTGCCCGACTTCCTGAAAGACGGCGAGGAGGAAACGG AGATTGG AGCATCTACCTCTCGGAAGAAAATATCCCAAATAATGCAGATACCAGTT? CCCTTCGGAA TCTGTAGGTTCTGftCGCGCCTTCCGAAGCGAAATACGACCTTGCCGAAATGTATCTCGAA ATCGGCGACCGCGATGCCGCTGCCGAGACAGTGCAGAAA1TGCTGGAAG3VAGCGGÍAGGC GACGrACXCAACGTGCCCAAGCATTGGCGCAGGAATTGGGTATTTGA NBM0341 Protein sequence MPAGRLPR CPMMTXPTDCTRSSR? QPPTH GYILKIWRQIKLIAASVAVAASPQAHAGL GGI.KIQSN DEPFSGSITVTGEEAKAL GGGSVTVSEKG TAKVH GDK? VIAVSSEQ? VRDPVLVFRIGAGAQVRE '/ TAILDPVGYSPKTKSALSDG AI THR APTAESQENQNA »RKTDKXDSANAAVKPAYl! LGKTHTVRKGEtvKQ? AW \ irpf (HLT EQVADAl.I? ÍCftNWlVSA HGRl.BAGSV HIPKLK? LKAE.PKPQT? KP ñETftSMPSEPS QATVEKPVSKPEAKVAA PEAKAEKPAVRPEPVPAANTA» VSETAAESAPQeAAASAIOTPTDETGNAVSEPVEQVSAE SETESG AGGGAA FDGLFGGSyT L L, L RLAQSKRARRTEESVPESEPDLDD7? DDGIE? TFAEVETPATPEPAPKNDVNDTLALDG ? SBEE SAKQTFDVETDTPSNRIDLDF DS SAAQNGILSGALTQDEETOKRADADíJNAIBSTDSVYEPETFNPYl? PVEIVIDTPEPE SVAQTAENKPE7VDTDFSDNLPSNNHIGTEETASAKPASPSGLAGF KRSSPETI EKTV AEVQTPEELHDF KVYE? DAVAETAPETPDFNAAADD SA LQPAEAPSVEENITETVAE TPDFNATADDLSAL QPSKVPAVEEIÜ? ETVADD SAL QPAEAPAVEENVTETVAETPD FNATADDLSALLQPSEAPAVEENAAETVADDLSA LQPAEAPAVEEKAAEIT ETPDS T SEADA PDFLKDGEEETVDWSIYIiSEEMIPNNADTSFPSESVGSDAPSEAKYDLAEMYLE IGDRDAAñETVQKLLEEABGDVLKRAQAI ^ AQELGI DNA sequence NMB0338 'ATGGñAAGGARCGGTGT? TTTGGTAAftAT? GTCGGCAATCGCATACtcCGTATG? CGrCC GAACACGCTGCCGCRTCCTATCCGAAACCGTGCAAATCGTT? AAACTAGCGCAATCTTGG TTCAGAGTGCGAAGCTGTCTGGGCGGCGTTTT? ATTTACGGAGOiAACATGftAACtTATC TATACCGTCATCAARATCATTATCCTGCTGCTCTTCCTGCTGCtTGCCGTCATTAATACG GATGCCGTTACCTTTTCCTACCTGCCG6GGCAAAAATTCGATTTGCCGCTGATTGTCGTA TGTTCGGCGCATTTGTAGTCGGTATTATTT7TGGAATGTTTGCCTTGTTCGGACGGTTG TTGTCGTTACGTGGCGAGAACGGC? GGTTGCGTGCCGAAGTAAAGAAAAATGCGCGTTTG ACGGGGAAGGAGCTGACCGC? CCACCGGCGCAAA? TTGCGCCCGAATCTACCAAACAGCCT NMB 0338 Protein sequence MERNGVFG IVGNRI R SSEHAAASVPKPCXSFKLAQSWFRVRSCLGGVFIYGANMKLI YTVI KI I IUJ FL LAVINTDAVTFSYLPGQKFD PLI WLFGAFWGI I FGMFALFGRL LSLRGENGR RAF? 'XKNARLTGKELTAPPAQNAPESTKQP Analysis of the polypeptide indicates that it is predicted to have two domains that extend along the membrane, residues 54 to 70 and 88 to 107. Thus, fragments from regions 1 to 53, and 108 to the extreme (C-terminal) can be particularly useful as immunogens.

NMB1345 DNA sequence ATGAAAAAACCTTTGATTTCGGTTGCGGCAGCA? GCTCGGCGTTGCTTTGG? CACGCC TATTATTTGGGTGTCAAAGCCGAAGAAAGCTTGACGCAGCAGCAAAAIAITAT GCAGGAA ACGGGCTTCTTGACCGTCGAATCGCACCAATATGÑGCGCGGCTGGTRTACCTCTATGGAA ACG? CGGTCATCCGTCTGAAACCCGAGTTGCTGAATAATGCCCGAAAATACC GCCGGAT AACCTGAAAACAGTGTTGGAACAGCCGGTTACGCTGGTTAACCATATCACGCACGGCCCT TTCGCCGGCGGATTCGGCACGCAGGCGXACATTGAAACCGAGTTCAAATACGCGCCTGAA ACGGAAAAAGTTCTGGAACGCTTTT? TGGAAAACAAGTCCCGGCTTCCCTTGCCAATACC GTTTATTTTAACGGCAGCGGTAAAATGGAAGTCAGTGTTCCCGCCTTCGATTATGAAGAG CTGTCGGGCATCAGGCTGCACTGGGAAGGCCTGACGGGAGAAACGGTTTATCAAAAAGGT TTCAAAAGC ACCGGAACGGCTATGATGCCCCCRRGTTTAAAARCAAGCTGGCAGACAAA GGCGATGC GCGTTTGAAAAAGTGCATGTCGATTCGGAAACTTCAGACGGCATC? ATCCG C? TGCTTTGGGCAGCAGCAATCTGACCTTGGAAAAATTCTCCC? AGAATGGAAAGAGGGT GTCGATTACAACGTCAAGTTAAACGAACTGGTCAATCTTGTTACCGATTTGCAGATTGGC GCGT? TATCAATCCCA? CGGCAGCA? CGCACCTTCCAAAATCGAAGTCGGCAAACTGGCT TTTTCAACCAAGACCGGGGAATCAGGCGCGTTTATCAACAGTGAAGGGCAGTTCCG T? CG? TACACTGGTGTACGGCGATG-AAAAATACGGCCCGCGGGACAGCCATATCGCTGCCGAA CACCTCGATGCTTCTGCC? TAACCG ATTGAAACGCA ^ GTTTGCACAAA ? TTCCGCC AAA AAAATGACCGAGGAÍÍCAAATCCGCAATGA? TTGA.? TGCCGCCGTCAAAGGAGAGGCTTCC GGACTGTTCACCMCAA? CCCGTAT GGACATTAAAACTTTCCGATGCACGCTGCCATCG GGAAAAATCGATGTGGGCGGAAAA? CATGTTTJIAAGACATGAAGAAGGAAG ?? TTGAAT CAATTGGGTTTGATGCTGAAGAAACCGAAGCCGACATCAGAATGAGTATTCCCCAAAAA ATGCTGGAAGACTTGGCGGT GTCAAGCAGGCAATATTTTCAGCGTCAATGCCGAAGAT GAGGCGGAAGGCAGGGCAAGTCTTGACGACATCAACGAGACCTTGCGCCTG? TGGTGGAC AGTACGG? TCAGAG ATGGCAAGGGAAAAATATCTGACTTTGAACGGCGACCAGATTGAT ACTGCCATTTCTCTG? ÜaAAACAATCAGTTGAARTTGA "iCGGTAAAACGTTGCAñAACG? A CCGG? GCC ATTTTGATGAAGGCGGTATGGTTTCAGAGCCGCAGC GTAA NMB 1345 Protein sequence MKKPLISVAAAL GVAT-GTPYYLGVKAEESLTQQQKIL ETGFLTVESHQY3RGWFTSME TTVIR KPEE.LNNARKY PDN TVLEQPVT VNHITHGPFAGGFGTQAYZETEFKYAPE TEKV ERFFGKQVPAS? O? TVYFNGSGKMEVSVPAFDYEE SGIR H EGLTGETVYQKG FKSYRNGYDAPLF IKLADKGDAAFEKVHFDSETSDGINPIA GSSNI.TLEKFS1EWKEG VDYNVKLHE VNLVTD QIGAFINPNGSIAPSKIEVGKLAFSTKTGESGAFINSEG FRF DTLVYGDEKYGPLDIfiIAAEHLDASALTVLKRKFAQISAK M? EEQIRNDLIAAVKGEAS Selection with vaccine sera Sera from the Meningococcal Reference Laboratory in Manchester have been available to the inventors. These sera have been developed from the OMV immunization of volunteers.

Mutants selected by Cl serum of vaccinated patients (selected once) The following sequences were isolated NMB0338 (as mentioned above) NMB0738 DNA sequence ATGA? GATCGTCCTGATTAGCGGCCTGTCCGGTTCGGGCAAGTCCG? CGCACTGCGCCAA ATGGAAGATTCGGGTTATTTCTGCGTGG? CAATTTGCCTTTGGA? ATGTTGCCCGCGCTG GTGTCGTATCA? ATCGAACGTGCGGACGAAACCGAATTGGCGGTCAGCGTCGATGTGCGT TCCGGCAtTGACATCGGACAGGCGCGGGAACAGATTGCCTCTCTGCGCAGACTGGGGCAC AGGGTTGA? GTTTTGTTTGTCGAGGCGGAAGAAAGCGTGTTGGTCCGCCGGTTTTCCGAA ACCAGGCGAGGACATCCTCTGAGCAATCAGGA? ATGACCTTGTTGGAAAGCTTAAAGAAA GAACGGGAATGGCTGTTCCCGCTTAAAGAAATCGCCTATTGTATCGACACTTCCAAGATG AATGCCCAACAGCTCCGC ATGCAGTCCGGCAGTGGCT AAGGTCGAACGTACCGGGCTG CTGGTGATTTTGGAGTCCTTCGGGTTC? AATACGGTGTGCCGAAC? ACGCGGATTTTATG TTCGATATGCGC? GCCTGCCCAACCCGTATTACGATCCCGAGTTGAGGCCTT? CACCGGT ATGGACAAGCCCGTTTGGGATTATTTG ACGGACAGCCGCTTGTGCAGGAAATGGTTGAC GACATCGAAAGGTTTGTTACGCATTGGTTACCGCGTTTGGAGGATGIA? GCAGGAGCTAC GTT? CCGTCGCCATCGGTTGCACGGGAGGACAGaACCGTTCGGTCTATATTGTCGAAAAA CTCGCCCGAAGGTTGAAA GGCGT? ATGAATTGCTGA ACGGCACAGACSGCGCAAAAC CTGTCAGACCGCTAA NMB0738 Protein sequence MKIV ISGLSGSGKSVALRQMEDSGYFCVDNLPÍ.EM PA VSYBIERADETEIAVSVDVR SGIDIGC? AREQIASLRR GHRVEVLFVEAEESVLVRRFSB'PEYÍGHPLSNQDMTL ESLKK EREW FP KEIAYCIDTS? INAQO RHAVRQW \ 'F.RTG VILESÍ GFKYGV? KN, ADFM FDMRSLPÍÍPYYDPELRPYTGMDKPVWDYLDGQPLV? JB 3VDDIERFVTH LFRLEDESRSY VFVAIGCTGGQHRSVYIVEKLA RL GRYE IRHRQAQNLSDR NMB0792 NadC family (transporter) DNA sequence ATGAACCTGCATGCAAAGGACAAAACCCAGCATCCCGAAAACGTCGAGCTGCTCAGTGCG CAGAAGCCGATTACCGACTTTAAGGGCCTGCTGACCACCATTATTTCCGCCGTCGTCTGT TTCGGCATTTACCACATCCTGCCTTACMCCCCGATGCCAATAAAGGTATCGCGCTGCTG ATTTTCGTTGCCGCACTTT6GTTTACCGAGGCCGTCCACATTACCGTAACCGCACTGATG GTGCCGATTCTCGCCGTCGTACTCGGTTTCCCCGACATGGACATCAAAA? GGCGATGGCT GATTTTTCCAACCCGATTATCTACATTTTTTTCGGCGGCTTCGCGCTTGCCACCGCCCTG CATATGCAGCGGCTGGACCGGAAAATCGCCGTCAGCCTGTTGCGCCTGTCGCGCGGCAAT ATGAAAGTGGCGGTTTTGATGTTGTTCCTCGTTACCGCCTTTCTGTCCATGTGGATCAGC AACACCGCCACCGCCGCGATGATGCTGCCTCTAGC? A GGGTATGCTGAGCCACCTCGAC CAGGAAAAAGAACACAAAACCTACGTCTTCCTCCTGCTCGGCATCGCCTATTGCGCCAGC ATCGGCGGCTTGGGCACGCTCGTCGGCTCGCCGCCCAACCTGATTGCCGCCAAAGCCCTA AATCTGGACTTCGTCGGCTGGATGAAGCTCGGCCTGCCGATGATGCTGTTGATTCTGCCC TTGATGCTGCTCTCCCTGTACGTCA? CCTCAAACCTAATTTGAACGAACGCGTGGAAATC? RAGCCGAATCCAtCCCTTGGACGCTGCACCGCGTGATCGCGCTGTTGATTTTCCTTGCC ACAGCCGCCGCGTGGATATTCAGCTCCAíAATCAAAACCGCCTTCGGCATTTCGAATCCC GACACCGTTATCGCCCTGAGTGCCGCCGTCGCCGTCGTCGTCTTCGGCGTGGCGCAATGG AAGGAAGTCGCCCGCAATACCGACTGGGGCGTGTTGATGCTCTTCGGCGGCGGCATCAGC CTGAGCACGCTGTTGAAAACATCCGGCGCGTCCGAAGCCTTGGGACAGCAGGTTGCCGCC ACCTTTTCCGGCGCGCCCGCATTTTTGGTGATACTCATCGTCGCCGCCTTCATTATTTTT CTSACCGAGTTCACCAGCAACACCGCCTCCGCCGCATTGCTTGTACCGATTTTCTCCGGC ATCGCTATGCAGATGGGGCTGCCCGAACAAGTCTTGGTATTCGTCATCGGCATCGGCGCA TCTTG7GCCTTCATGCTGCCGGTTGCCACACCGCCTAACGCGATTGTGTTCGGCACGGGC TTAATCAAGCAACGCGAAATGATGAATGTCGGCATACTGCTGACATCCTCTGCGTAGTA? TTGGTTGCTCTGTGGGCTTATGCTGTACTGATGTAA NMB0792 Protein sequence NLHAKDKTQHPENVE LSAQ PI7DFKG LTTI ISAWCFGIYBILPYSPDANKGIALL I F VAA ?? AVH I TVTA M V P I W W FPDM Ül KKAMADFSNPI? And I F? G GFALATAL HMQ LD KIA L S GNMKVAVLM TAFLSI «IIS» TATAA 3MIJ? LA ^ iG! Ll SHLD QEKEHKTYVF LLGIAYCASlGGIíGTLVGSPPNLIAAKALHLDFVGWMK GLPMM IL? LMLLS h Y V I KPNLN2RVE? KAES 1 P TLHRVIAL I FLATAAAW 1 FS S Kl KTAFG I SN P DTVIALSAAVAVVVFGVAQ KEVAR TDWGVLM FGGGISLSTL KTSGA3EALGQ VAA TFSGAPAFliVILIVAAFIIFLTEFTSKTASAA LVPlFSGIAMQMGLPEQV VFVIGIGA SCAF LP ATPPNAIVFGTGLIKOREMMN GI NILCW ALWAYAVL d NMB0279 DNA sequence ATGC? ACG? CAAATCAAACTGAAAAATTGGCTTCAGACCGTTTATCCCGAACGGGACTTC GATCTGACTTTTGCGGCGGCGGATGCTGATTTCCGCCGCTATTTCCGTGCAACGTTL'TCA GACGGCAGCAGTGTCGTCTGCATGGJLTGC? CCGCCCGACAAGATGAGTGTCGCACCTTAT TTGAAAGTGCAGAAACTGTTTGACATGGTCAATGTGCCGCAGG3ATTGCACSCGGACACG GATCTGGGGTTTGTGGTATTGAACGACTTGGGCAATACGACGTTTTTGACCGCAATGCTT CAGGAACAGGGCGAAACGGCGCACAAAGCCCTGCTGTTGGAGGCAATCGGCGAGTTGGTC GA TTGC ^ JJAAGGCGAGCCGTGAAGGGGTTTTGCCCGAATATGACCGTGAAACGATGTTG CGCGAAATCAACCTGTTCCCGGAATGGT? TGTCGCAAAGAAT'IGGGGCGCGAATTAACA TTCAAACAACGCCAACTTTGGCAGCAAACCGTCGATACGCTGCTGCCGCCCCTGTTGGCG CAGCCC? AAGTCTATGTGCACCGCGACTTTATCGTCCGCAACCTGATGCTGACGCGCGGC AGGCCGGGCGTTTTAGACTTCCAAGACGCGCTTTACGGCCCGATTTCCTACGATTTGGTG TCGCTGTTGCGCGATGCCTTTATCGAATGGGAAGAAGA? TTTGTCTTGGAC? TGGTTATC CGCTACTGGGAAAAGGCGCGGGCTGCCGGCTTGCCCGTCCCCGAAGCGTTTGACGAGTTT TACCGCTGGTTCGAATGG? TGGGCGTGCAGCGGCACTTGAAGGTTGCAGGCATCTTCG A CGCCTG ACT? CCGCGACGGCAAAGACA? ATACCGTCCGGAAATCCCGCGTTTCTTAAAC TATCTGCGCCGCGTATCGCGCCGTTATGCCGAACTCGCCCCGCTCTACGCGCTCTTGGTC GAACTGGTCGGCGATGAAGAACTGGAAACGGGCTTTACGTTTTAA NMB0279 Protein sequence MQRQIKLKFF? ÍWTVYPERDFDLTFAAADADF'RRYFRATFSDGSSVVCMDAPPDKMSVAPY LKVQKLFDMVWVPQVKADTDLGFVVL? ÍDLGKTTFLTALÁL E GETAHKALLLEAIGELV ELQKASRBGV PEYDRETMLRE? NLGPEÍ'ÍFVAKSLGRELTFKQR L QQVDTLLPPLLA QPKVYVHRDFIVRNLMLTRGRPGVLDF DALYGPISYDLVSLLRDAFIEHEEEPV1.DLVI RYWEKAPAA.GLPVPSAFDEFYRKFEWMGVQRE KVAGIFARLYYRDGKDKYRPEIPRFLN YLRRVSRRYAELAPLYALLVELVGDEELETGF F NMB2050 DNA sequence ATGGAACTGATGACTGTTTTGCTGCCTTTGGCGC? CGTTGGTGTCGGGCGTGTTGTTTACA TGGTTGC? GATGAAGGGCCGGTTTC? GGGCGAGTTTGCCGGTTTGñACGCGCACCTGGCG GAAAAGGCGGCAAG? TGTGATTTTGTCGAACAGGCACACGGCAAAACCGTGTCGGAATTG GCGG GACGGGAAATACCGGCATTTGCAGGHCGAAAATTATGCt TGGGCAACCGT TTTTCCGCAGCCGAAAAGCAGATTGCCCATTTGCAGGAAAAAGAGGCGGAGTCGGCGCGG CTGAAGCAGTCGTATATCGAGTTGCAGGAAAAGGCACAGGGTTTGGCGGTTGAAAACGAA CGTTTGG-CAACGCAGCTCGGACAGGAAGGGAAGGCGTTTGCCGACCAATATGCCTTGGAA CGCCAAATCCGCCAAAGAATCGAAACCGATTTGGAAGAAAGCCGCCAAACTGTCCGCGAC GTGCAAARICGACCTTTCCGATG CGGCÍACCGTTTTGCCGCAGCCGAAAAACAGA'PTGCC CATTTGCAGGAAAAAGGGCGGAAGCGGAGCGGTTGAGGCAGTCGCATACCGAGTTGCAG GAAAAGGCACAGGGTTTGGCGGT GAAAACGAACGTTTGGCAACGCAAATCGAACAGGAA CGCCTTGCTTCTGAAGAGAAGCTGTCCTTGCTGGGCGAGGCGCGCAAAGTTTGAGCGA? GTTTCAAAATCTTGCCAACACGATTTTGGAAGAAAAAAGCCGCCGTTTTACCGAGCAG AACCGCGAGCAGCTCCATCAGGTTTTGAACCCGCTAAACGAACGCA? CCACGGTTTCGGC GAGTTGGTCAAGCAAACCTATGATAAAGAATCGCGCGAGCGGCTGACGTTGGAAAACGAA TTGAAACGGCTTCAGGGGTTGAACGCGCAGCTGCACAGCGAGGCAAAGGCCCTGACC2VAC GCGCTGACCGGTACGCAGAATAAGGTTCAGGGCAATTGGGGCGAGAGGATTCTGGAAACG G? TTTGGAAAA? RCCGGCCTTCAGAAAGGGCGGGAATATGTGGTTCAGGCGGCATCCGTC CGAAAAGAGGAAGACGGCGGCACGCGCCGCCTCCAGCCCGACGTTTT6GTCAACCTGCCC GACAACAAGCAGATTGTGATTGATTCCAAGGTCTCGCTGACAGCTTATGTGCGCTACJICG CAGGCGGCGGATGCGGATACGGCGGCACGCGAACTGGCGGCACACGTTGCCAGCATCCGT GCACACATGAAAGGCTTGTCGCTGAAGGATTACACCGATTTGGAAGGTGTGJUiiCACATTG GATTTCGTCTTT? TGTTTATCCCTGTCGAACCGGCCTACCTGTTGGCGTTGCAGAATGAC GCGGGCTTGTTCCAAGAGTGTTTCGACAAACGGATTATGCTGGTCGGCCCCAGTACGCTG CTGGCGACTGGGAGGACGGTGGCGAATATTTGGCGCAACGAACAGCAAAATCAGAACGCA CTGGCGATTGCGGACGAAGGCGGCAAGCTGTACGACAAGTTTGTCGGCTTCGTACAGACG CTCGAAAGCGTCGGCAA? GGCATCGATCAGGCGCAAAGCAGTTTTCAGCGGCATTCAAG r-ftarTTG CGA GßGCGCGGGAATCTGGTCGGACGCGCCGAGAACTGCGTCTGTTGGGC GTGAAGGCAGGCAAACAACTTCAACGGGAGTTGGTCGAGCGTTCCAATGAAACAAOGGCG TTGTCGGAATCTTTGGAA ACGCGGCAGAAGATGAAGCAGTCTGA NMB2050 protein sequence KELMTVL PLAAI? FSGV.FTWl. ^ RGRQGEFAGLNAHLAE AARCDFVEQAUGKTVSSL? V DGKYRHLQDENYALGNRFSAAEKQIAHLQEKEAESARLKQSYIELQEKAQG Vese RL? TQLGQER AFADQYALERQIRQRIETDLSESRQTV PVQNDLSD GNRr? AASKQIA HLQEKEAEAERLRQSHTELQEKAQGLA ^ NER? IATQIEQERLASEEKLSLLGEARKSLSD OFQNIANTILEEKSRRFTEQNREQLHQVLNPLMERIHGFGSLVKQTYDKESRERLTLENE LKRLQGLNAQLHSEA ALTNALTGTQNKVQGNWGEMILETVLBNSGLQ GREWVQAASV RKEEDGGTRRLQPDVLVNLPDMKQIVIDSKVSIJTAYVRYTOAADADTAARE AAHVASIR AHM GI? SLKDYTDLEGWTLDFVFMFRPVF.PAYLLALQNDAGLFQECFDKRLMLVGPSTIJ J? TLRTVAHIWRHEQQNQNALAIADEGGKLYDKFVGFVQTLESVGKGIDQAQSSFQTAFK QLASGKGHLVGRAEKLRI.LGVKAGKQLQRDLVERS " ETTALSES EYA EDEAV NMB1335 CreA protein DNA sequence ATGAACAGACTGCTACTGCTGTCTGCCGCCGTCCTGCTGAC7GCCTGCGGCAGCGGCGAA ACCGATAAAATCGGACGGGCAAGTACCGTTTTCAACATACTGGGCAAAAACGACCGTATC GAAGTGGAAGGATTCGACGATCCCGACGTTCAAGGGGTTGCCTGTTATATTTCGTATGCA AAAAAAGGCGGCTTGAAGGAAATGGTCAATTTGGAAGAGGACGCGTCCGACGOVTCGGTT TCGTGCGTT AGACGGCATCTTCGATTTCTT TGACGAAACCGCCGTGCGCAAACCG? AA GAAGTRTTCAAAICACGGTGCGAGCTTCGCGTTCAAGAGCCGGCAGATTGTCCGTTATTAC GACCCCAAACGCAAAACCGTCGCCTATTTGGTGTACAGCGATAAAATCATCCAAGGCTCG CCGAAAAATTCCTTAAGCGCGGTTTCCTGTTTCGGCGGCGGCATACCGCAAACCGATGG3 GTGCAAGCCGATACTTCCGGCAACCTGCTTGCCGGCGCCTGCATGATTTCCAACCCGATA GAAAATCTCGACAAACGCTGA NMB 1335 Protein sequence MNRLLLLSAAVLLTACGSGETDKIGRASTVFNILGKNDR? EIVEGFDDPDVQGVACYIS A KGGLSEMVNLEeDASDASVSCVQTAS ISFDETAVRKPKBVFKHGAS AFKSRQIVRYY DPKRKTFAYLVYSDKIIQGSPK-NSLSAVSCF6GGIFQTDGVQADTSGNLLAG? CMISNPI ENLDKR NMB2035 DNA sequence ATGACCGCCTTTGTCCACACCCTTTCAGACGGCATGGAACTGACCGTCGAAATCAAGCGC CGTGCCAAGAAAA? CCTGATTATCCGCCCCGCCGGCACACA '? ACCG-TCCGCATCAGCGTC CCACCCTGCTTCTCCGTCTCCGCTCTAAACCGCTGGCTGTATGAAAACGAAGCCGTCCTG CGGCAAACACTGGCGAAAACACCGCCGCCGCAAACTGCCGAAAACCGGCTGCCCGAATCC ATCCTCTTCCACGGCAGACAGCTTGCCCTCACCGCCCATCAAGACACGCAAATCCTGCTG ATGCCGTCTGAAATCCGTGTTCCCGAAGGCGCACCCGAAAAACAGCTTGCGCTGCTGCGG G? CTGTTTGGAACGGCAGGCGCACAGTTACCTGATTCCCCGCCTCGAACGCCACGCCCGC ACCACACAACTGTTCCCCGCCTCCTCCTCGCTGACCTCTGCCAAAACCTTC GGGGCSTG TGCCGCAAAACC? CAGGCA? ACGCTTCAACTGGCGGCTGGTCGGCGCACCGGAATACGTT GCCGACT.ATGTCTGCA7ACACGAACTCTGCCACCTCGCCCATCCCGACCACAGCCCCGCC TTTTGGGAACTGACCCGCCGCTTCGCCCCCTACACGCCCAAAGCGAAACAGTGGCTCAAA ATCCACGGCAGGGAACTTTTCGCCTTAGGCTGA NMB2035 Protein sequence MTAFVHTLSDGMELTVEI KRRAKICNLI IRPAGTHTVRISVPPCFSVSALNR LYBNEAVL RQTLAKTPPPQTAENRLPESILFKGRQLALTAEQDTQILLMPSEIRVPEGAPEKQLALLR DFLERQAHSYLIPRLERHARTTQLFPASSSLTSAKTFWGVCRKTTGIRFN RLVGAPEYV AD VC I H ELC HLAH PDH S PAFWE TRRFAP YT P AKQWLKI HG RSLFALG NMB 1351 protein Fmu and F v DNA sequence ATGAACGCCGCACAACTCGACCATACCGCCAAAGTTTTGGCTGAAATGCTGACT? TCAAA CAGCCTGCCGATGCCGTCCTCTCCGCCTATTTCCGCGAACACAAAAAGCTCGGCAGTCAA GATCGCCACGAAATCGCCGAAACCGCCTTTGCCGCGCTGCGCCACTATCAAAAAATCAGT ACCGCCCTACGCCGTCCGCACGCGCAGCCGCGCAAAGCCGCTCTCGCCGCACTGGTTCTC GGCAGAAGCACC? ACATCAGCCA? ATCAAAGACCTGCTTGATGAAGAAGW? CAGCGTTC CTCGGCAATTRGAAAGCCCGTAAAACCGAGTTTTCAGACAGCCTGAATACCGCCGCAGAA TTGCC? CAATGGCTGGTGGAACAACTGAAACAGCATTGGCGCGAAGAAGAAATCCTCGCT TTCGGCCGCAGCATCAACCAGCCTGCCCCGCTCGACATCCGCGTCAA.CACTTT6AAAGGC AAACGCGATAAAGTGCTGCCGCTGTTGCAAGCCGAAAGT6CCGATGCAGAGGCAACGCCT TATTCGCCTTGGGGO? TCCGCCTGAJ? AACAAAATCGC6CTTAACAAAC? CGAACTGTTT TTAGACGGCACACTGGAAGTCCAAGACGAAGGCAGCCAGCTGCTTGCCTTATTGGTGGGC GCAAAACGAGGCGAAATCATTGTCGATTTCTGTGCCGGTGCCGGCGGTAAAACCTTGGC7 G? CGGTGCGCAAATGGCGAACAAAGGCAGAATCTACGCCTTCGATATCGCCGAAAAACGC CTTGCCAACCRCAAACCGCGTATGACCCGCGCCGGACTGACCAATATCCRCCCCGAACGC ATCGGCAGCGAACACGATGCCCGTATCGCCCGACTGGCAGGCAAAGCCGACCGTGTGTTG GTGG? CGCGCCCTGCTCCGGTTTGGGCACTTTACGCCGCAATCCCGACCTC? AATACCGC CAATCCGCCGAAACCGTCGCCAACCTTTTGGAACAGC? ACACAGCATCCTCGATGCCGCC TCCAAACTGGTAAAACCGCAAGGACGTTTGGTGTACGCCACTTGCAGCATCCTGCCCGAA GAAAACGAGCGGCAAGTCGAACGTTTCCTGTCCGWVCATCCCGAATTTGAACCCGTCAAC TGCGCCGAAC? GCTTGCCGGTTTGAAAATCGATTTGGATACCGGC? ATACCTGCGCCTC AACTCCGCCCGACACCAAACCGACGGCTTCTTCGCCGCCGTATTGCAACGCAAATAA NMB 1351 protein sequence MNAAQLDHTAKVLREMLTFKQPADAVLSAYFREH KLGSQDRHEIAET? FAALRHYQKIS TALRRPHAQPRKAAIAALVLGRSTNI SQ KDLLDEEETAFLGNLKARKTEFS DSLNTAAS PQBLVEQLKQH REEEI LAFGRSINQPAPLDI RVNTLKGKRDKVLPLLQAESADAEAT P YS PÍ '? GIRLKNKIALNKHELFLDGTLEVQDEGSQLLALLVGAKRGEI I VDFCAGAGGKTLA VGAQ ^ AN GRIYAFDIAEKRLA. LKPRH7RAGLTKLHPER? GSEHDARIARLAGK? P L VDAPCSGLGTLRRNPDLKYRQSAETVAHILEQQHSILDAASKLVKPQGRLVYATCSILPE EWELQVERFLSEHPEGEPVNCAELLAGLKSDLDTGKYLRLNSARHQTDGFFAAVLQR NMB 1574 IlvC DNA sequence ATGCAAGTCTATTACGATAAAGATGCCGATCTGTCCCTAATCAAAGGCAA? ACCGTTGCC ATCATCGGTTACGGGTCGCAAGGTCATGCCCATGCCGCCAACCTGAAAGATTCGGGTGT? AACGTGGTGA'PTGGTCTGCGCCAAGGTTCTTCTTGG AAA? AGCCGAAGCAGCCGGTCAT GTCGTCAAAACCGTTSCTGAAGCGACCAAAGAAGCCGA? GTCGTTATGCTGCTGCTGCCT GACGAAACCAGGCCTGCCGTCTATCACGCCGAAGTTACAGCC? A? TTGAAAGAAGGCGCA ACGCTGGCATGTGCACACGGCTTCAACGTGCACTACAACCAAATCGTTCCGCGTGCCGAC TTGGACG GAGTATGGTTGCCCCCA AGGTCCGGGCCATACCGTACGCAGTGAATACAAA CGCGGCGGCGGCGTGCCTTCTCTGATI CCGTTTACCAAGAC &??? ATTCCGGCAAAGCCAAA GACATCGCCCGGTCTTATGCGGCTGCCAACGGCGGCACCATAGGCGGTGTGATTGAAACC ACTTTCCGCGAAGAAACCGAAACCGATCTGTTCGGCGA CAAGCCGTATTGTGCGGCGGC GTGGTCGAGTTGATCAAGGCGGGTTT GAAACCCTGACCGAAGCCGGTTACGCGCCTGAA ATGGCTTACTGCGAATGTCTGCACGAAATGAAACTGATCGTTGACCTGATTTTCGAAGGC? GGTATTGCCAATATGJÍACTACTCCATTTCCA, \ CAATGCGGAGTACGGCGAATACGT ACC ATT CVACCGGCGAATACGCAAUIATGTTTATCCAAGAGGGTAATGTCAACTATGCGTCTATG GGCCCTGAGPGGrCAATGCTTCCAGCAAAGAAGCCATGCGCAATGCCCTGAAACG ACTGCCCGCCGCCGTCTGAATGCCGACCACCAAGTTGAAAAAGTCGGCGCACAACTGCGT GCCATGA? GCCTTGGATTACTGCCAACAAATTGGTTGACCAAGACAAAAACTGA NMB1574 protein sequence MQVYYDKDADLSLIKGKTVAIIG GSQGHAHAAMLKDSGVNWIGLRQGSSWKKAEAAGH VVKTVÑEATK? ADVVMLLLPDETMPAVYHAEVTANLKEGAT? JAFAHGFNVKYNQIVPRAD LDVIMVAPKGPGKTVRSEYKRGGGVPSLIAVYQDKSG AKDIALSYAAANGGTKGGVIET TFREETETDLFGSQAVLCGGVVELIKAGFETLTEAGYAPEMAYFECLHEKKLIVDLIFEG GIAN NYSISNH? EYGEYVTGPEVVNASSKEAMRNALKRIQTGEYAKMF? QEGNVNYASM TARRRLNADHQVEKVGAQLRAMMPWITANKLVDQDKN NMB1298 rsuA DNA sequence ATGAAACTTATCAAATACCTGCAATATCAAGGCATAGGAAGCCGCAAGCAGTGCCAATGG CTGATTGCCGGCGGTTAGGTTTTCATCAACGGAACCTGCATGGACGACACCGATGCAGAC CCC ATCGATTCCTCA'Í'CCGTCGAAACGTGGGATATI'GACGGGGAAGCAGTAACCGTCGG GAACCCT? TTTCTACATCATGCTCAACAAGCCTGAAGATTACGAAACTTCGCACAAACCC AGCACTACCGCAGCGTATTCAGCCYGTTCCCCGACAATATGCGGA? CATCGATAGGCAG GCGGTCGGCAGGCTGGATGC? GATACGACCGGCGTATTGCTGATTACCAACGACGGCA? A CTGAACCACAGCCTGACTTCGCCGAGCAGAAÍÍAATTCCCAAGCTGTACGAAGTAACGCTC AAAC.B.CCCCACAGGAG AACGCTCTGCGW'ACCTTGAAAJVACGGCGTGCTGCTCC.ACGAC GAAA? CGAAACCGTTTGTGCCGCCCATGCCGTTT? GAAAAACCCGACCACCCTGCTGCTG ACCATTACCGAAGGAAAATACCACCA GTCAAACGCATGATCGCCGCCGCCGGCWÍCCGC CACCTTCATCGCCGGCGAT GTGC ^ ^ CGCACATCTGGAAACAGAAAACCTC.GIAACCCGGG GAATGGA? ATTTATCGAB TGTCCAAAATTCTGA NMB 1298 Protein sequence MKLJKYLQYQGIGSRK C0WLIAGG? VFINGTCMDDTDAD? D3SSVETLDI DGEAVTVVP? .PY YI ^ Jl.NK? E? ETSKKPKH S FS FP SM NI DMQAVG DADTTGVl? ITWDGK LN H S LTS PSRKI P L YE VT LKH PTGF-T I -CETLK 'G VLLK DF.NET VCAA AV KNP T TLLL TI TEGKYHQVKW-il ^ AAGNRVQHLHRRRFAHLETENL PGEWKFIECPKF NMB1856 Family Lys R (transcription regulator) DNA sequence ATGPAAACCAATTC? GAAGA? CTGACCGTATTTGTTOL GTGGTGGAAAGCGGCAGCTTC AGCCGTGCGGCGGAGCAGTTGGCGATGGCAAATTCTGCCGTA ^? GCCGCATCGTCAAACGG CTGG? GGAAAAGTTGGGTGTGAACCTGCTCAACCGCACCACGCGGCAACTCAGTC GACG G? AGAAGGCGCGCAATATTTCCGCCGCGCGCAGAGAATCCTGCAAGAAATGGCAGCGGCG GAAACCGAAATGCTGGCAGTGCACGAAATACCGCAAGGCGTGTTGAGCGTGGATTCCGCG ATGCCGATGGTGCTGCATCTGCTGGCGCCGCTGGCAGCAAAATTCAACGAACGCTATCCG C? TATCCGACTTTCGCTCGTTGCTTCCGA? GGCTATATCAATCTGATTGAACGCAAGTC GATATTGCCTTACGGGCCGGAGAATTGGACGATTCCGGGCGGCGTGCACGCCATCTGTTT GACAGCCGCTTCCGCGTAATCGCCAGTCCTGAATACCTGGCAAAACACGGCACGCCGCAA TCTACAGA? GAGCTTGCCGGCCACC? ATGTTTAGGCTTCACCGAACCCGGTTCTCTAAAT ACATGGGCGGTTTTAGATGCGCAGGGAAATCCCTATAAGATTTCACCGCAC'FTTACCGCC AGCAGCGGTGAAATCTTACGCTCGTTGTGCCTTTCAGGTTGCGGTATTGTTTGCTTATCA GATTTTTTGGTTGA AACGACATCGCTGAAGGAAAGTTAATTCCCCTGCTCGCCGAACAA ACCTCCGATAAAACACACCCCTTTJVATGCTGTTTATTACAGCGATAAASCCGTCAATCTC CGCTTACGCGTATTTTTGGATTTTTTAGTGGAGGAACTGGGAAACAATCTCTGTGGATAA NMB1856 protein sequence MKTNSEELTVFVQVVESGSFSRAAJÍQLAJ4RNSAVSRIVKRLEEKLGVNLLNRTTRQLSLT EEGA YFRRAQRILQEMAAAETEMLAVHEIPQGVLSVDSAMPMVLHLLAPLAAKFNERYP HIRL3LVSSEGY? MLIERKVDIALRAGELDDSGLRARHLFDSRFRVIASPEYLAKHGTPQ STEELAGHQCLGFTEPGSLNTWAVLD? QGNPYKrSPHFTASSGEILRSLCLSGCGIVCLS DFLVDNDIASGKLIPIíLAEQTSDKTHPFNAVYYSDKAVI? LRLRVFLDFLVEELGNNLCG NMB0119 A.TGA7GAAGGATTTGAATT7GAGCAACAGCCTGTTCAAAGGCTACAACGACAAACA DNA sequence GGC TTA? TGTTTGTGGCTATGAATGGGGTTGGAGTAAAGCCG? TGAGGCTGCTTATGTAGCA GG7GAATAC? AACTCCCTGAAAiCAAAATCGñCCATACA? TTGCAAACAAATCCCTC AT TTCGGAGAGCAGGCAAAAAAGTGGCGTTACGACAATACGATAAA? AATTGGTTTGAAATG TGGGGACACCCCTTAGACG? A? ATGGATTGGGCGGTGCATTTGAAAAATCCCTGGTTCAA ACCAAC'J'GGGCTGCTACACAGGGCAIC? CTATCGACAATCCCGACAAGTTCACACAACCC GAGCACATCGATAATTTTCTCTACCAC? TCGAAAAACTGCGTCCGAAAGTCATCCTCTTC ATGGGCAGCAGGtTGGCGGATTTTCTGAACAACCAAAATGTACTGCCACGCTTCGAGCAG TTGGTCGGTAAGCAGACCAAACCGCTGGAGACGGTGCAAAAAGAATTTGACGGTAC? TTCA CGT? TGTCAAATTCCAATCGTTTGAAGATTGCGAAGTCGTCTGCTTTCCCCATCCCAGT GCC? GTCGCGGTCTATCTTACGATTACA? CGCCTTGTTTGCGCCTGAíiATGAACCGGATT TTATCGGACTTTí ? AACAACACGCGGATTCAAATAA NMB01 19 Protein Sequence ^ iMKDIrfLSK FKGYrKDKHG SICGYE ^ KG53KAD? ^ JlYV GE K PEN IDHTF l) KS and FGEQA KWRYDNTIKNtfFE W HPLDENGLGGAFSKSLVQTRHAATQGNGIDMPDKFTOP ER? DMFLYHIEKLRP VILFMGSRLADFLNSQHVLPRFEQLVGK TKPLETVQKEFDGTP. FKVKFQSFEDC? WCFPHPSASRG SYDY1AX.FAPEM RILSDFKTTRGFK NMB1705 rfaK DNA sequence ATGGAAAAAGAATTCAGGATATTAAATATCGTA CGGCCAAGATTTGGGGTGGAGGCGAA CAATATGTCTATGATGTTTCAAAAGCATTGGGGCTTCGGGGCTGCACAATGTTTACCGCC GTCA? TAAAAATATGAATTGATGCACAGGCGATTTTCCGAAGTTTCTTCCGTTTTCACA. ACGCGCCTTCACACGCTCAACGGGCTGTTT CGCTCTACGCACTTACCCGCTTTATCCGG AAAAACCGCAT? TCCCACCTGATGATACACACCGGCAAAATTGCCGCCTTATCCATACTT TTG.AAAAACTGACCGGGGTGCGCCTGATATTTGTCAARCATAATGTCGTCGCCAACAAA ACCGATTTTTACCACCGCCTGATACAGAAAAACACAGACCGCTTTATTTGCGTTTCCCGG CTGGTTTACG? TGTGCAAACCGCCGACAATCCCTTTAAAGAAAAATACCGGATTGTTCAT AACGGTATCGATACCGGCCGTTTCCCTCCCTCTC? AGAAAAACCCGAC? GCCGTTTTTTT ACCGTCGCCTACGCCGGCAGGATCAGTCCAGAAPVAGGATTGGAAAACCTGATTGAAGCC TGTGTGAT? CTGCATCGGAAATATCCTCAAATCAGGCTCAAATTGGCAGGGGACGGACAT CCGGATTATATGTGCCGCCTGA ^ GCGGGACGT? TCTGCTTCAGGAGCAGAACCATTTGTT TCTTTTGAX'IGGGTTTACCGAAAAACTTGCTTCGTTTTACCGCCAAAGCG? TGTCGTGGTT TTGCCCAGCCTCGTCCCGGAGGCATTCGGTTTGTC? TTATGCGAGGC ATGTACTGCCGA ACGGCGGTGATGTCCAATACTTTGGGGGCGCAAAAGGAAATTGGCGAACATCATCAATCG GGGATTCTGC? GGACAGGCTGACACCTGAATCTTTGGCGGACGAAATCG? ACGCCTCGTC TTGAACCCTGAAACGAAAAACGCACTGGCAACGGCAGCTCATCAATGCGTCGCCGCCCGT TTTACCATCAACCATACCGCCGACAAATTATTGGATGCAATATAA NMB1705 Protein sequence MEKEFRILNIVSAEaBGGGEQYVYDVSKALGLRGCTMFTAVNKNNSLMHRRFSEVSSVFT TRLHTLNGLFSLYAL7RFIRKNS1SHLMIHTGKIAALSILLKK TGVRLIFVKHNVVAM TDFYHRLIQKNTDRF? CVSRLVYDVQTADBPFKEKYRIVHNGIDTGPFPPSQ? KPDSRFF? VAYAGRISPEXGLENLIEACVI HR YPQ? RLKLAGDGHPDY CRLKRDVSA3GAEP ^ V SFEGFTEK ASFYR SDVWL? SLVPEAFGLSLCE MYCRT VISKTI, GAQKF.1VEH.HQS GILLDRLTPESLADEIERLVLNPET íiALRTAAHQCVAARFTINHTADKLLDAI NMB2065 protein DNA sequence ATGCAGGAACAGAATCGGAAACCAAGTTTTCCCATAGTGATGTTGCTGGTGTCGGTTGCC HemK CTGTGGATAGCGTCTTTATCCAATGTTGCATTTÍATTTGGGCAATC? TGGAAGCATGGAG GGTTTGACCGTTTTGATTTTGGGGTCGATATTTGCTTCTTTGGATATCAGGTATTGTGCG GTC? ATGCGAATTATGTT? GGTTGGCGGCCATTGTTTTGCTGGCGTTGCGGAAGAAGGTC GTGCCTGGCCATGCGGCACTTTGGGGCTTGGCGTTGGTGGCTTTCAGTGTGAAAGCCGTA TACGTCGATGAAGCAGGGAATACATCGGATATTGTGCGCTACGGTGCAGGATTTTATTTG TGGTATGCCGCATTTGCGGTTGCCACCATCGGTACGTTTGCCGGAAAÍÍ? ATAAGGAAAGA AAAGCCGCATCAGCGGCAGACGGGATAAAAATGACGTTTGATAAA.TGGTTGGGCTTGTCA AAACTGCCTAAAAATGAAGCAAGAATGCTGCTACAATATGTTTCGGAATATACGCGCGTG CAGTTGTTGACGCGGGGCGGGGAAGAATGCCGGACGAAGTCCGACAGCGGGCGGACAGG CTGGCGCAACGCCGTC7GAACGGCGAGCCGGTTGCCTATATTTTAGGTGTGCGCGAATTT? ATGGCAGACGCTTTACAGTCAATCCGAGCGTGCTGATTCCGCGCCCCGAAACCGAACAT TTGGTCGAAGCCGTATTGGCGCGCCTGCCCGAAAACGGGCGCGTGTGGGATTTGGGGACG GGCAGCGGCGCGGTTGCCGTAACCGTCGCGCTCGAACGCCCCGATGCGTTTGTGCGCGCA "PCCGACATCAGCCCGCCCGCCCTTGAAACGGCGCGGAAAAATGCGGCGGATTTGGGCGCG CGGGTCAAATTTGCACACGGTTCGTGGTTCGACACCGATATGCCGTCTGAAGGGAAATGG GACATCATCGTG'PCCAACCCGCCCTATATCGAA? ACGGCGATAAACATTTGTTGCAAGGC GATTTGCGGTTTGAGCCGCAAATCGCGCTGACCGACTTTTCAGACGGCC AAGCTGCATC CGCACCTTGGCGCAAGGCGCGCCCGACCGTTTGGCGGAAGGCGGTTTTTTATTGCTGGAA CACGGTTGCGATCAGGGCGCGGCGGTGCGCGGCGTGTTGGCGGAGAATGGTTTTTCAGGA GTGGA? ACCCTGCCGGATTTGGCGGGTTTGGACAGGGTTACGCTGGGGAAGTATATGAAG NMB2065 Protein sequence MQSQNRKPSFPIVHLLVSVALKIASLSNVAFYLGWHGSMEGLTV'LILGSI FASLDIRYGA VYASY \ TOlJ? XVLLALRKKV rPV? JAA W6 LVAFSVKAVYVDEAGNTSDIVRYGAGFYL WYAAFAVATIGTFAGKS ERKAASAADGII < MTFDKWLGLSKLPKNEARMLLQYVSEYTRV? J LTRGGEEMPDEVRORADRLAQRRLNGEPVAYILGVREFYGRRFTVKPSVLIPRPETEH LVSAVLARLPSWGRVWDLGTGSGAVAVTVALERPDAFVRASDI SPPALETARKNAADIíGA RVSFAnGS FDrD PSEGK DIIVSNPPYlENGDKP-LLQGDLRFEPQrALTDFSDGLSCI RTL ?? 3APDRIAEGGFLLLEHGFDQGAAVRGV AENGFSGVETLPDLAGLDRVTLGKYMK HLK Mutants selected by the 17 D serum of the vaccinated (selected only once) NMB0339 DNA sequence ATGGACAACGAATTGTGGATTATCCTGCTGCCGATTATCCGTTTGCCCGTCTTCTTCGCG ATGGGCTGGT? TGCCGCCCGCGTGG? TATGAAAACCGTATTGAAGCAGGCAAAAAGCATC CCTTCGGGARTRTATAAAAGCTTGGACGCTTTGGTCGACCGCAACAGCGG? CGCGCGGCA ACGGAGTTGGCGGAAGTCGTCGACGGCCGGCCGCAATCGTATGATTTGAACCTCACECTC GGC3UU4CTTT? CCGCCAGCGTGGCGAAAACGACAAAGCCATCAACATACACCG6ACAATG CTCGATTCTCCCGATACGGTCGGCGAAÑAGCGCGCGCGCGTCCTGT? TGAATTGGCGCAA AACTACCAAAGTGCGGGGTTGGTCGATCGTGCCGA CAGATTTTTTTGGGGCT ^? CAAGAC GGTAAAATGGCGCGTGAAGCCAGACAGCACCTGCTCAATATCTACCAACAGGACAGGGAT TGGGAAAAAGCGGTTGAAACCGCCCGGCTGCTCAGCCATGACGATCAGACCTATCAGTTT GAAATCGCCCAGTTTTATTGCGAACTTGCCCAAGCCGCGCTGTTCAAGTCCAATTTCGAT GTCGCGCGTTTCAATGTCGGCAAGGÍ CTCGAAGCCÑACAAAFIA ^? 'GCACCCGCGCCAAC ATGATTTTGGGCGACATCGAACACCGACAAGGCAATTTCCCTGCCGCCGTCGAAGCC AT GCCGCCATCGAGCAGCAAAACCATGCATACTTGAGCATGGTCGGCGAGAAGCTTTACGAA GCCTATGCCGC5CAGGGAAAACCTGAAGAAGGCTTGAACCGTCTGACAGGATATATGCAG ACGTTTCCCGAACTTGACCTGATCAATGTCGTGTACGAGAAATCCCTGCTGCTTAAGTGC G? GAAAGAAGCCGCGCAAACCGCCGTCGAGCTTGTCCGCCGCAAGCCCGACCTT AAC? GC GTGTACCGCCTGCTCGGTTTGAAACTCAGCGATATGAATCCGGCTTGGAAAGCCGATGCC GACATGATGCGTTCGGTTA.TCGGACGGCA6CTACAGCGCAGCGTGA.TGTACCGTTGCCGC AACTGCCACTTCAAATCCCAAGTCTTTTTCTGGCACTGCCCCGCCTG? ACAAATGGCAG ACGTTTACCCCGAATA? AATCGAAGTTTAA NMB0339 Protein sequence MDNELWI ILLPIILLFVFFAMG? 'IFAARVDKKTV QAKSIPSGFYKSLDALVDRWSGRAA RELAEWDGRPQSYDLNLTLG LYRQRGENDKAIHIHRTMLDSPDTVGE RARVLFELAQ NYQSAGLVDRAEQ? FLGLQDGKaAREARQHLLNIYQQDRDWEKAVETARIiLSHDDQTYQF The AQF And CELAQ AALFKS N FDVARFN VGXALEAN KKCTRANMI LGDi EHRQGN FPAA VE AY AAIEQQNHAYLSMVGEKLYEAYAAQGKPEEGLKRLTGYMQTFPELDLINWYEKSLLLKC EKEAAQTAVELV ^ KPDLKGVYRLLGLKLSDMNPAWKADADMbSRSVIGSQL RSV YRCR NCHFKSQVFFWHCPACNKWQTFTPNKIEV Selection with patient serum The inventors have a collection of sera from patients with acute disease and convalescent patients available for selection. This is from individuals infected with different serogroups of N. meningitides. The selections have been carried out with sera from patients with acute disease (A) or convalescent patients (C). The period between the acute infection and the collection of the sera was from two weeks to three months.

NMB0401 p? TA DNA sequence ATGTTTCATTTTGCATTTCCGGCACAA? CTGCCCTGCGCCAAGCSATAACCGATGCCT? C CGCCGTAATGAAATCG? AGCCGTACAGGATATGTTGCAACGTGCACAGATGAGCGACGAA GAGCGCA = CGCCGCCTCCGAGCTTGCCCGCCGTTTGGTTACCCAAGTCCGCGCCGGCCGC ACCAAAGCCGGCGGCGTGGATGCGCTGATGCACGAGTTTTC? CTCTCCAGCGAAGAAGGC ATCGCGCTGATGTGTCTCGCAGAAGCCCTGCTGCGTATCCCCGACAACGCCACGCGCGAC CGCCTGATTGCCGACA? GATTTCAGACGGCAACTGGAAAAGCCATTTGAACAACAGCCCT TCCCTCTTCGTCAATGCTGCCGCCTGGGGCCTGCTGATTACCGGCAAACTGACCGCCACA AACGACAAACAAA7GAGTTCCGCACTCAGCCGCCTGATCAGCAAAGGCGGCGCACCGCTC ATCCGCCAAGGCGTAAATTACGCCATGCGGCTTCTGGGCAAACAGTTCGTAACCGGACAG ACCATTGAAGA? GCCCTGCAAAACGGCAAAGAACGCGAAAAAATGGGCTACCGCTTCTCC TTCGATATGTTGGGCGAAGCCGCCTACACCCAAGCCGATGCCGACCGCTACTACCGCGAC TATGTCGAAGCCATCCACGCCATCGGCAAAGATGCGGCAGGACAAGGCGTTTACGAAGGT AACGGTATTTCCGTCAAACTTTCCGCCATCCATCCGCGCTACTCGCGCACCCAACACGGC CGCGTGATGGGCGAACTGTTGCCGCGCCTGAAAGAGCTGTTCCTTTTGGGTAAAAAATAC GATATCGGTATCAACATCGATGCCGAAGAAGCCAACCGTCTGGAGCTGTCTTTGGATTTG ATGGAGGCTTTGGTTTCA? ACCCTGACTTGGCTGGCTACAAAGGTATCGGRTTC GTTGTC CAAGCCTACCAAAAACGTTGTCCGTTCGTTATCGACTACC? GATCGACCTTGCCCGCCGC AACAACCAAAAACTAATG? TCCGCCTCGTCAAAGGCGCGTATTGGGACAGCGAAATCAAA TGGGCGCAAGTGGACGGCTTGACGGCTATCCGACCTACACCCGCAAAGTCCACACCG? C ATCTCCTACCTCGCCTGCGCGCGCAAACTGCTTGCCGCGCAAGACGCGGTATTCCCGCAA TTTGCCACCCACAACGCCTACACTTTGGGCGCAATCTACCAAATGGGTAI \ AGGCAAAGAT TTTCAACACCAATGCCTGCACGGTATGGGCGAAACCCTGTACGACCAAGTCGTCGGCCCG CAAAACTTAGGCCGCCGCGTGCGCG7GTACGCCCCAGTCGGCACAC? CGAAACCCTGCTC GCCTACTTGGTGCGCCGCCTGTTGGAAAACGGCGCGAACTCGTC7TTC TCAACCAAATC GTCGATGAAAACATCAGCATCGACACGCTCA7CCGCAGCCCGTTCGACACCATCGCCGAA CA GGCATCCACCTGCACAACGCCCTGCCGCTGCCGCGCGATTTGTACGGCAAFTTGCCGT CTGAACTCGCAAGGCGTGGACTTGAGCA? CGAAAACGTATTGCAGCAGCTTCA? GAACAG ATGAACAAAGCCGCCGCGCAAGACTTCCACGCCGCATCCATCGTCAACGGCAAAGCCCGC GATGTCGGCGAAGCGCAACCGATTAAAAACCCTGCCGACCACGACGACATCGTCGGCACA GTCAGCTTTGCCGA.TGCCGCGCTTGCCCAAGAAGCGGTTGGCGCAGCCGTTGCCGCG TC CCCGAATGGAGTGCGACACCTGCCGCCGAACGCGCCGCCTGCCTGCGCCGTTTTGCCGAT TTGCTGGAGCAGCACACCCCAGCACTGATGATGCTTGCCGTGCGCGA? G-AGGCAAAACG CTGAACA CGCCATT GCCGAAGTGCGC AAGCCGTCGATTTCTGCCGCTACTACGCAAAC GAAGCCGAACATACCCTGCCTCAAGACGC? AAAGCCGTCGGCGCGATTGTCGCCATCAGC CCGTGGAACT? CCCGCTCGCCATCTTTACCGGCGAAGTCGTTTCCGCATTGGCGGCAGGC AAC? GCC? CATCGCCAAACCCGCCGAACAA CCAGCC ^? GATTGCCGGTTATGCCGTTTCC CTCATGCACGAAGCCGGCATCCCGACTTCCGCCCTGCAACTCGTCCTCGGCGCAGGCGAC GTGGGTGCGGCATTGACCAACGATGCCCGCATCGGCGGCGTGATTTTCACCGGCTCGACC GAAGTGGCGCGCCTGATC? ACAAAGCCCTTGCCAAACGCGGCGACAATCCCGTCCTGATT GCCGAAACCGGCGGACAAAACGCCATGATTGTCGATCCACCGdCTTGCCGAGCAAG? C TGCGCCGACGTATTGAACTCCGCCTTCGACAGCGCGGGACAACGCTGCTCCGCCCTGCGC ATTITGTGCGTCCAAGAAGACG7TGCCGACCGTATGCTCGACATGATCAAAGGCGCTATG GACGAACTCGTCGTCGGCAAACCGATTCAGCTCACTACCGATGTCGGCCCCGTCA? CGAT GCCGAAGCACAGCAAAACCTGTTGAACCACATCAACAAAATGAAAGGTGTTGCCAAGTCC TACCACG.AAGTCAAAACCGCCGCCGA7GTCGATTCCAAAAAATCCACGTTCGTTCGCCCC ATCCTGTTTGAATTGAACAACCTCAACGAACTGCAACGCGAAGTCTTCGGTCCCGTCCTG CACGTCGTCCGCTACCGCGCCGACGAACTCGACAACGTCATCGACCAAATCAACAGCAAA GGCTACGCCCTGACCCACGGCGTACACAGCCGCATCGAAGGCACGGTACGCCACATCCGC AGCCGCATCGAAGCCGGCAACGTTTACGTCAACCGCAACATCGTCGGCGCAGTCGTCGGC GTACAGCCCTTCGGCGGACACGGTCTGTCCGGCACAGGCCCCAAAGCAGGCGGTTCGTTC TACCTGCAAAAACTGACCCGCGCCGGCGAATGGGTTGCCCCGACCCÍGAGCCAAATCGGA CAGGCGGACGAAGCCGCACTCAAACGCCTCGAAGCACTGGTTCACAAACTACCGTTCAAC GCCGAAGAGAAAAAAßCCGCAGCGGCCGCTTTGGGACACGCCCGCATCCGCACCCTGCGC CGTGCCGAAACCGTCCTTACCGGACCGACCGGCGAGCGCAACAGCATCTCATGGCACGCG CCCAAACGCGTTTGGATACACGGCGGCAGCACGGTTCAAGCCTT7GCCGCACTGACCGAA CTTGCCGCCTCCGGCATACAGGCAGTGGTCGAACCCGA AGCCCCTTGGCTTCCTACACT GCCGAGTTGGAAGGTCTGCTGCTGGTCAACGGCAAACCCGAAACCGCCGGCATCAGCCAC GACTGA NMB0401 Protein sequence HF'IFAFP? TALRQAITDAYRR? ÍEG EAVQDH QRAQ. SDEERWA ^ ELARRLVTQVRAGR T KAGG VDALMHE FSI. S SEE G I ALMC LAE ALL R? DK AT RDRL? AD IS DG1TO KS HLWN SPS FV [51 AA? WGLLI TGKLTAT K DKQMSS LS RLI S KGG APPL RQG VN YAMRLLGKQ FVT GQ TLEEAIIQNGKERE MGYRFSFDMLGEA? YTQADADRYYRDYVEAIHA? GKDAAGQGVYEG I GI SVKIASAIRPRYSATQHGRVMGELLPRLKELFLLGKKYDIGINIDASEANRLELSLDL MBALVSDPDLAGYKGIGFVVQAYQKRCPFVI DY IDLARRNNQKLM? RLVKGAYWDSEIK WAQVDGLNGYPTYTRKVHTDI SYLACARKLLSAQDAVFPQFATHS AYTLGAIYQMGKGKD FSHQCLHGMGETLYTX? VVGPQNI? RRVRVYAPVGTHETLIAYLVRRLLEKGAKSSFVNQI VDENT SI DTLI RSPFDTIAEQGIHLHNALPLPRDLYG CRLNSQ6VDLSNENVLQQLQEQ MNKAAA DFHAASIVNGKARDVGEAQPIKNPADRDDIVGTVSFADAALAQEAVGAAVAAF PEWSATPAAERAACLRRPADLLEQHT9ALMMLAVREAG TLNSALAEVREAVDFCRYYAN 5AEH7I.PQ? KAVGAI VAI S PWNFPLAI FTGEVVSALAAGNTVJAKPAEQTSLIAGYAVS MHEAGIPTS? LQLVLGAGDVGAA THDARIGGVIFTGSTEVAR I ^ ALAKRGDNPVLI AETGGQNAMIVDSTAL? EQVCADVLNSAFDSAGQRCSALRILCVQEDVADRMLDMIKGAM DELW6KPI LTTDVGPVI3AEAQQNLLKHINKMKGVAKSYHEVKTAADVDSKKS7FVRP ILFELNKLNEIIQREVFGPVLHWRYRADEL? NVIDQINSKGYALTHGVHSRIEGTVRHIR S RX EAGNVYVNRN? VG AWGVQ PFGGHGLSGTG PKAGGS FYLQK TRAGEWVAPTLSQI QADEAAL R EALVH LPFHAEEKKRAAAALGHARIRTLRRAETVI, TGPTG? RNSI SWHA PKRVWIHGGSTVQAFAALTELAASGIQAVVRPDSPLASYTADLEGLLLVNGKPETAGJ SH VAALS PLDSARKQELAAH DGALIRI PSENGLDI QVFEEISCSV TTAAGGMASLMAVA D NMB1335 CreA Previously provided DNA and protein sequences NMB1467 PPX DNA sequence ATGACCACCACCCCCGCAAACGTCCTCGCCTCCGTCGATTTGGGTTCC / VACAGTTTCCGC CTCCGATTTGCGAAAC? ACARCGGACAATTAAAAGTCATCGATTCGTTCAAACAGATG GTGCGCRTCGCCGCCGGACTGGACGAACAGAAAAATCTGAGTGCCGCTTCCCAAGAACAG GCTTTGGACTGTCTGGCAAAATTCGGCGAACGCCTGCGCGGCTTCCGCCCTGAACAGGTA CGCGCCGTGGCAACCAACACATTCCGCGTTGCCAAAAACATCGCAGATTTCCTTCCCAAA GCCGAAGCGGCATTGGGTTTCCCCATCGAAATCATCGCCGGGCGCGAAGAGGCGCGGCTG ATTTATACCGGCGTGATCCACACCCTCCCCCCGGGCGGCGGCAAAA? GCTGGTTATCGAC ATCGGCGGCGGTTCGACAGAATTTGTCATCGGCTCGAC6CTGAATCCCGACATTACCGAA AGCCTGCCCTTGGGCTGCGTAACCTACAGCCTGCGCTTCTTCCAAAACAAAATCACCGCC AAAGACTTCCAATCTGCCATTTCCGCCGCCCGCAACGAAATCCAGCGTATCAGCAAAAAT? T AGGCGCGAAGGTTGGGATTTCGCCGTCGGCACATCGGGTTCGGCAAAATCCARCCGC GACGTGCTTGCCGCCGAAATGCCCCAAGAGGCGGACATTACCTACAAAGGCATGCGCGCC CTCGCCGAACGCATCATCGAAGCCGGTTCGGTCAAAAAAGCCAAATTTGAAAACCTGAAA CCGGAACGCATCGAAGTTTTTGCCGGCGGACTTGCCGTGATGATGGCGGCGTTTGAGGAA ATGAAACTCGACAGGATGACCGTAACCGAAGCCGCCCTGCGCGACGGCGTGTTTTACGAT TTGATCGGGCGCGGTTTAAACGAAGA'FATGCGCGGACAAACGGTTGCCGAGTTCC AACAC CGCTACCACGTCAGCCTCAATCAGGCGAAACGCACCGCCGAGACCGCGCAAACCTTTATG GACAGCCTCTGCCACGCTAAAAACGTTACAGTTCAAGAGCTTGCCTTGTGGCAACAGTAT CTCGGACGCGCCGCCGCGCTGCACGAAATCGGTTTGGACATCGCCCACACCGGCTATCAC AAGC? TTCCGCCTACATCCTCGAAAACGCCGATATGCCGGGTTTCTCACGCAA? GAACAG ACCATACTTGCCCAACTGGTCA7CGGTCATCGCGGCGATATGAAAAAAATGAGCGGCATC ATCGGCACCA? CGAAATGGTGTGGTATGCCGTTTTGTCCCTGCGCCTTGCCGCACTGTTC TGCCGTTCGCGCCAAGACCTGTCTTTCCCGAAAAA TGCAGTTGCGCACGGATACGGAA ?? AGCTGCGGCTTCATCCTGCGTATTGACAGGGAATGGCTGGAACGCCATCCCCTGATTGCC GACGCATTGGAATATGAAAGCGTCCAATGGCA AAAATCAATATGCCGTTCAAAGTCGAG GCCGTCTGA NMB 1467 protein sequence MTTTPAhJVLASVDLGSNSFRLQICENNNGOLKVIDSF QMVRFAAGLDEQKNLSAASQEQ ALDCLAKFGERLRGFRPEQVRAVATNTFRVAKííIADFLPKAEAALGFPIEI? AGREEARL? YTGVIRTLPPGGGKH VIDIGGGSTEFVIGSTLMPDITESÍ.PLGCVTYSLRFFONKITA KD FUS AI I S A RWE QXI 5 KNMRREGWDFAVG TS AKS G S I DV? AAASM PQEADI T KGMRA I.AERIIEAGSVKKA FENLKPERIEVFA6GLAVMMAAFEEMKLDRMT \ 'TEAALRDGVFYD I.? GRGLN E DMRGQT V AE FQHR Yf! V SLNQAKR? AE T AQT FM DS LCK A N VT VQELALWQQ Y L G R AA H E I GL DI AH? G Y K KH S Y I ENADMP G S RKEQT I AQL I GG R KKMS G I IGTNEM WYA ^ SLRLAALFCRSRQDLSFPKNMQLRTDTESCGFILR? DRSWLBRHPLIA DALSYESVQWQKINMPFKVEAV NMB2056 Hcm A? GAACGGTA ATACTACTACGGCACAGGCCGCCSCAAAAGTTCAGTGGCTCGTGTATTC CTGATTX ^? A-AGGTACAGGTCAAATCATCGTAAACGGTCGTCCCGTTGACGAATTCTTCGCA CGGG.A CCAGCCGA ^. ^ TGGTTGTTCGCCAACCCTTGGTTCTGACTGAAAACGCCGAATCT T? C ACATCAA? GTCAATGTTGTTGGCGGCGGCGAAACCGGCCAGTCCGGCGCAATCCGC CACGGCA7TACCCGTGCCCTGATCG? CTTCGATGCCGCG7TG.? ACCCGCCTTG7CTCAA GCTGGTTTTGTTACCCGCGATGCCCGCGAAGTCGÍiACGTAAAAAACCGGGTCTGCGCAAA GC? CGCCGTGCAAAACAATTCTCC A CGTTAA NMB2056 Protein sequence MKíKYYYGTGRRKSSVARVFLI GTGQIIVNGRPVDEFFARETSRMWROPLVi? 'EK'AES I-X1I ^ NVVGGGETGQ5GAIRHG.T? RAT.IDFD? JILKPALSQAGF \ 'TRDAREV? R KPGLR ARRAKQFSKR NMB0808 DNA sequence? TGTCCGCCCTCC? CCCCATCA7CAACCGCCTGA? TCTGC ?? AAGCCCGGACAGCCGCTCG GAACTTGCCGCCTTTGCAGGCAAACACTGACCCTGAACATTGCCGGGCTGAATlCTGGCG GGriCGCATCACGGftAGACGG? TTGCTC7CGGCGGGAAACGGCTTTGCAGACACCGAAATT ACCTTCCGC? ACAGCGCGGTA (^ 67iAAATCCTCCAAGGAGGCGAACCCGGGGCGGGCGAC ATCGGGC7CGAAGGCGACCTCATCCTCGGCA7CGCGGTACTGTCCCTGCTCGGCAGCCTG CGTTCCCGCGCATCGGACGAAT? GGCACGGA7TTTCGGCACGCAGGCAGACA? CGGCAGC CGTGCCGCCGACATCGGACACGGCA7CAAACAAATCGGCAGGAACATCGCCGACAAA? C GGCGGATTTTCCCGCG? -lTCCGAGTCCGCAAACATCGGC.? IACGAAGCCCTTGCCGACTGC CTCGACGAAATAGCAGACTGCGCGACGGCG7GGAACGCCTCAACGAACGCCTCGACCGG CTCGAACGCG CAT7TGGATAGACTAA NMB0808 Protein Sequence MSALLPIIBRLILQSPDSRSELAAFAGKTLTLNIAGLKLAGRITBDGLLSAGNGFADTEI TFRNSAVQKII.QGGEPGAGDIGLEGDLILGIAVLSLLGSIASRASDE ARIFGTQADIGS RAADIGHGI QIGRNIAEQIGGFSRESESASIGHEALADCLDBISRLRDGV5RLNERLDR LERDIWID NMB0774 upp DNA sequence ATGAACGTTAATGTTATCAACCATCCGCTCGTCCGCGftCAAATTAACCCTGATGAGGGAG GCGGATTGCAGCACCTACAAATTCCGGACGCTTGCCACCGAGCTGGCGCGCCTGATGGCA TACGAGGC ^ ÜIGCCGTGATTTTGAAATCGAAAAATACCTTATCGACGGATGGTGCGGTCAG ATTGAAGGCGACCGCATCAAGGGCAAAACATTGACCGTCGTTCCCATACTGCGTGCAGGT TTGGGTATGCTTGACGGTGTGCTCGACC? GATTCCGACTGCCAAAATCAGTGTAGTCGGA CTGCAGCGCGACGAAGAAACGCTGAAGCCTATTTCCTATTTTGAGAAATTTGTGGACAGT ATGGACGAACGTCCGSCTTTGAT? ATCGATCCTATGCTGGCGACAGGCGGTTCGATGGTT GCCACCATCGACCTTTTGAAAGCCAAGGGCTGCAAAAATA.TCAAGGCACTGGTGCTGGTT GCCGCGCCCGAGGGTSTGAAGGCGGTCA ^ CGACGCGCACCCTGACGTTACGAT? TACACC GCCGCGCTCGACAGCCACTTGAACGAGAACGGCTACATCATCCCCGGCTTGGGCGA? GCG GGCGACAAGATTTTCGGCACGCGCTAA NMB0774 protein sequence KSVNVINHPLVRHKLTUÍREADCSTYKFRTLATELARLMAYEASRDFE EKYLJDGWCGQ IEGDRIKGKTLTWP LRAGLGMLDGVTJDLIPTAKISWGLQRDEETLKPISYFEKFVDS MDER ALIlD taATGGS &?!?! IVATIDLLKAKGCKNlKALVLVA7 \ PSGVKAV IDAHPDVTIYT AALDSHLNENGYSIPGLGDAGDK1 GTR? NMA0078 putative integral membrane protein DNA sequence TTGGCGTTTACTTTAATGCGTCGCGCCATGA.TACGTAAAATGCCCTATACGGAAGATATG CGCCCAGGCGATACCGCTAATCCTTATGGTGCGTCCAA? IGCGATGGTGGAACGGATGTTA ACCGACATCCAAAAAGCCGATCCGCGCTGGAGCATGATTTTGTTGCGTTATTTCAATCCG AT7GGC6CGCATGAA ?? GCGGCTTGATTGGCGAGCAGCCAAACGGaArCCCGAATAVi ',' TG 7TGCCTTATATCTGCC? AGTGGCGGCAGGCAAACTGCCGCAATTGGCGGTATTTGGCGAT GACTACCCTACCCCCGACGGCACGGGGATGCGTGACTATATTCATGTGATGGATTTGGCA G.AAGGCCATGTCGCGGC7ATGCAGGC.AAAAAGTAATGTAGCAGGCACGCATTTGCTGAAC T7AGGCTCCGGCCGCGCTTCTTCGGTGTTGG? AATCATCCGCGCA7TTGAAGCAGCTTCG GGTTTGACGATTCCGT? TGAAGTCAAACCGCGCCGTGCCGGTGATTTGGCGTGCTTCTAT GCCGACCCTTCCTATACAAAG3CGCAAATCGGCTGGC? AACCCAGCGTGATTTAACCCAA ATGATGGAAGACTCATGGCGCTGGGTSAGT? AT? ATCCGAATGGCTACGACGATTAA NMA0078 Protein sequence ^! ^ TLMR AMT KM? 7EDÍí? GDT N? GASCAM E M TDIQK DPPWSf'SIL RY NP IGAH? SGLLGEQPNGIPFFNLLPYICQVAAG LPQLAVFGDDYPTPDGrGMRDYIRVMÜLA EGKVAAM AKSK'VAGTHLLNLGSGRASSVLE? IRAFEAASGLTIPYEVKPRRAGDLACFY ADPSYTKAQIGWQTQRDLTQ2ÍMSD5KR VSKNPNGYDD NMB0337 amino acid transferase branched chain DNA sequence ATGAGCAGACCCGTACCCGCCGTATTCGGCAGCGTTTTTCACAGTCAAATGCCCGTCCTC GCCTACCGCGAAGGCAAATGGCAGCCGACCGAATGGCAATCTTCCaAAGACCTCTCCCTC GCACCGGGCGCGCACGCCCTGCACTACGGCAGCGAATGTTTCGAGGGACTGAAAGCCTTC CGTCAGGCAGACGGCAAAATCGTGCTGTTCCGTCCGACTGCCAATATCGCGCGTATGCGG CAAAGTGCGGACA? TTTGCACCTGCCGCGCCCCGAAACCGAAGCTTATCTTGACGCGCTA? TCAAATTGGTCAAACGTGCCGCCGATGA? ATTCCCGATGCGCCTGCCGCCCTGTACCTG CGTCCGACCTTAATCGGTACCGATCCCGTTA7CGGC? AGGCCGGT7CTCCTTCCGAAACC GCCCTGCTGTATATTTTGGCTTCCCCCGTCGGCGACTATTTCAAAGTCGGATCGCCCGTC AAAATTTTGGTGGAAACCGAACACATCCGCTGCGCCCCGCAT? 7GGGCCGCGTCAAATGC GGCGGCAACTACGCTTCCGCCATGCACTGGGTGCTGAAGGCGAAAGCCGAATATaSCGCA AATCAAGTCCTGTTCTGCCCGAACGGCGACGTGCAGGAAACCGGCGCGTCCAACTTTATC CTGATTAACGGCGATGAAATCATTACCAfiACCGCTGACCGACGAGTTTTTGCACGGCGTA ACCCGCGATTCCGTACTGACGGTTGCCftAAGATTTGGGCTATACCGTCAGCGAACGCAAT TTCACGGTTGACGAACTCAAAGCTGCGGTGGAAAACGGTGCGGAAGCCATTTTGACCGGT ACGGCAGCCGTCATCTCGCCCGTTACTTCCT7C6TCATCGGCGGCAAAGAAA7CGAAGTG AAAAGCCAAGAACGCGGCTATGCCATCCGTAAGGCGATTACCGACATCCAGTA7GGTTTG GCGG AGACAAAT CGGCTGGC TGGTTGAAGTCTGCTGA NMB0337 Protein sequence MSRPVPAVFGSVFHSQMPVLAYREGKWQPTEWQSSQDLSIAPGAHALHYGSECFEGLKAF R ADGKIVLFRPTANIARMRQSADILHLFSPETEAYLDALIKLVKRAADErPDAPAALYL RPTLIGTDPVIGKAGSPSETALLYILASPVGDYFEC7GSPVKILVETEHIRCAPHP5GRVKC GGNYASAMHWVLIÍAKAEYGANQVLFCPNGDVQETGASNFIL? WGDEIITKPLTDEFLHGV TRDSVLTVAKDLGYTVSERNFTVDELKAAVENGAEAILTGTAAVISPVTSF ^ IGGKEIEV KSQERGY? IRKATTDIQYGLAEDKYGWLV? VC NMB0191 family of protein to DNA sequence ATGAGTGCGAAC TCCTTGCCATCGCCAATCAGAAGGGCGGTG GGGCAAAACGACGACG ACGGTAAATTTGGCGGCGTCGCTGGCATCGCGCGGCAAACGCGTGCTGGTGGTCG? TTTG GATCCGCAGGGCAATGCGACGACGGGCAGCGGC? TCGACAAGGCGGGTTTGCAGTCCGGC GTTTATCAGGTC? TATTGGGCGATGCGGACGTGCAGTCGGCGGCGGTACGCAGC? AAGAG GGCGGATACGCTGTGTTGGGTGCGAACCGCGCGCTGGCCGGCGCGGAATCGAACTGGTG CAGGAAA7CGCCCGGGAAGTGCGT7TGA? AAACGCGCTCAAGGCAGTGGAAGAAGATTAC GACTTTA7CCTGATCGACTGCCCGCCTTCGCTGACGCTGTTGACGCTTAACGGGCTGGTG C5CGGCGGGCGGCGTGATTGTGCCGATGTTGTGCGAATATTACGCGCTGGAAGGGATTTCC GATRRGATTGCG? CCGTGCGCAAAATCCGTCAGGCGGTCAATCCCGATTTGGACATCACG GGT \ TCGTGCGCACGATGTACGACAGCCGCAGCAGGCTG? TTGCCGAAGTCAGCGAACAG TTGCGCAGCCATTTCGGGGATTTGCTTTTTGAAACCGTCATCCCGCGCAATATCCGCCTT GCGGAAGCGCCGAGCCACGGTATGCCGGTGATGGCTTACGACGCGCAGGCAAAGGGTACC? AGGCGTATCTTGCCTTGGCGGACGAGCTGGCGGCGAGGGTGTCGGGGAAATAG NMB0191 protein sequence SANIIAIAHQKGGVGIWTTTVNIAASLASRGXRV VVDLDPQGNATTGSG? DKAGLQSG VYQVLLGt DVOSA?, RSKF.GGYA \ rLGANRALASAEIELVOEIAREVRLKNALKAVEBDY DFILIDCPPSLTLLTLSGLVAAGGVIVPMLCEYYALEGISDLIATVRKIRQAVNPDLDIT Gl? TlTMYDSRSRLVAEVSEQLRSHFGDLLFETVIPRNIRLAEAPSHGMPVMAYDAQAKGT KAYLALADELAARVSG NMB1710 Giutamato dehydrogenase (gdhA) DNA sequence ATGACTGACCTGAACACCCTGTTTGCCAACCTCAAACAACGCAATCCCAATCAGGAGCCG TTCCATCAGGCGGRTGAAGAAGTCTT? TGAGTCTCGATCCGTTTTTGGCAAAAAATCCG AAATACACCCAGCAAAGCCTGCTGG.AACGCATCGTCGAACCCGAACGCGTCG7GATGTTC CGCGTAACC? GGCAGGACGATAAAGGGCAAGTCCAAGTCAACCGGGGCTACCGCGTGCAA ATGAGTTCCGCCATCGGTCCTTACAAAGGCGGCCTGCGCGTCCATCCGACCG? CGATTTG GGCGTATTGAAATTCCTCGCTTTTGAACAAGTGTTCAAAAACGCCTTGACCACCCGGCCT ATGGGCGGCGGCAA / VGGCGGTTCCGACTTCGACCCCAAAGGCAAATCCGATGCCGAAGTA ATGCGCTTCTGCCAAGCCTTTATGACCGAACTCTACCGCCACATCGGCGCGGACACCGAT GTTCCGGCCGGCGACATCGGCGTAGGCGGGCGCGAAATCGGCTACCTGTTCGGACAATAC .AAAAAAATCCGCAACGÍÍ.GTTTTCTTCCGTCCTGACCGGCAAAGGTTTGGAATGGGGCGGC AGCCTCATCCGTCCCGAAGCGACCGGCTACGGCTGCGTCTATTTCGCCCAAGCGATGCTG C AAACCCGC AACGAT GTT TTGAAGGC AAACGCG CCTG T T T GTG CCGGCTCCGGCAAT GCGCAATACGCCGCCGAAAAAGCCATCCAACTGGGTGCGAAAtíTACTGACCGTTTCCGAC TCC7 ACGGCTTCGTCCTCTTCCCCGACAGCGGTATGACCGAAGCGCAACTCGCCGCCTTG ATCGAAT7GAAAGAAGTCCGCCGCGAACGCGTTGCCACCTACGCCAAAGAGCAAGGTCTG CAATACTTTGAAAAACAAAAACCGTGGGGCGTCGCCGCCGAAAL'CGCCCTGCCCTGCGCG ACCCAGAACGAATTGGACGAAGAAGCCGCCAAAACCCTGTTGGCAAACGGCTGCTACGTC G? TGCCGAAGGTGCGAARATGCCGTCGACTTTGGGCGCGGTCGAGCAATTTAT AAAGCC GGCATCCTCTACGCCCCGGGAAAAGCCTCCAATGCCGGCGGCGTGGCAACTTCAGGTTTG GAAA7GAGCCAAAACGCCATCCGCCTGTCTTGGACTCGTGAAGAAGTCGACCAACGCCTG 7TCGGCATCATGCAAAGCATCCACGAATCC GTCTGAAATACGGCAAAGTCGGCGACACA TAAACTACGTCAATGGTGCGAACATTGCCGGTGTCGTCAAAGT? GCCGATGCGATGCTG GC3CAAGGCTTCTAA NMB1710 protein sequence MTDLSTLFAKLKQRNPNQEPFHQAVEBVF SLDPFLAKNPKYTQQSLLERIVSPERVVMF RVTWQDDKGQVQVNRGyRVQMSSAIGPY GGLRFHPTVDLGVLKFLAF? QVFKtíALTTLP MGGGKGGSDFDPKGKSDASVMRFCQAFMTELYRHIGADTDVPAGDIGVGGREIGYLFGQY KKIRNEFSSVLTGKGLEWGGSLIRPEATGYGCVYFAOAWLQTRHDSFEGXRVLISGSGt? ' AQYAAEKAIQLGAKVLTVSDStWFVLFPDSGMTEAQLAALIELKEVRRERVATY? KEQGL QYFEKQKP GVAASIALPCATQNBLDEEAAXTLLANGCYWAEGANMPSTLGAVEQFIECA GILYAPGKASNAGGVATSGLEMSQNAIRLS TREEVDORLFGIKQSIHESCL YGKVGDT VKY VÑG iAGrvVADAMLAQGF NMB0062 gl uc o sa-1-phosphate thymidyltransferase (rfbA-1) DNA sequence ATGAAAGGCATCATACTGGCAGGCGGCAGCGG CGCGCCTC ^? ACCCCATCACGCGCGGC GTATCCAAACAGCTCCTGCCCGTGTACGACAAACCGATGATTTATTACCCCTTGTCGGTT TTGATGCTGGCGGGAATCCGCGATATTTT6GTGATTACCGCGCCTGAAGACAACGCCTCT TTC? AACGCCTGCTTGGCGACGGCAGCGArTTCGGCAT'fTCCATCAGTTATGCCGTG AA CCCAGTCCGGACCGCTTGGCACAGGCATTTP-TCATCGGCGAAGAATTTATCGGCAACGA.C AATGTTTGCTTGGTTTTGGGCGAC? ATATTTTTTACGGTCAGTCGTTTACGCAAACATTG AA? CAG3CGGCAGCGCAAACGCACGGCGCAACCGTGTTTGCTTATCAGGTCAAAAACCCC GAACGTTTCGGCGTGGTTGAATTTAACGAAAAC? TCCGCGCCGTTTCCATCGAAGAAAAA CCGCA? CGGCCC.AAATCCGATTGGGCGGTAACCGGCTTGTATTTCTACGACAACCGCGCC GAG GTC T AAAC TCGCC AGCTCAAACCG T CCG CACGCGGCG AATTGG AAAT T TC ACCGACC AACCGGATGTATTTG6AAGACGGCTCGCTCTCCGTTCAAATATTGGGACGCGGTTTCGCG TGGCTGGACACCGGCACCCACGAGAGCCTGCACGAAGCCGC? TCATTCGTCCAA? CCGTG CAAAATATCCAAAACCTGCACATCGCCTGCCTCGAAGAAATCGCTTGGCGC? ACGGTTGG CTTTCCGATGAAAí iCTGGA ^ TTG CTG T TA GAATTGGCGCGCCCGATGGCGAAAAACCAATACGGCCAA CGC CT GTTG AAAAA ATAR NMB0062 Protein sequence KKGI ILAGGSGTR YPITRGVSKQLLPVYDKPMIYYPLSVL? 4LAGIRDILV? TAPSDNAS FKRLLGDCISDFGISISYAVQPS? DGLAQAFI1GEEFIGMDWVCLVLGD «IFYGQSFTC > TL KQ? AAQ7HG T FA YQVXNPERFGW E FNSN FRAV S I EEKPQR KS DWA VTG YFY DNR? VEFAKQLKPSARGELEITDL? -iRMYLEDGSLSVQILGRGFAMLDTGTHESLHEAASFVQTV QNIONLHJACLeeJAHRNGIíLSDEKLEELARPMAKNQYGeYLLRLLKK NMB 1583 Imldazolgticerol-phosphate dehydratase (hisB) DNA sequence ATGAATTTGACTAAAACACAACGCCAACTGl ^ aWlCTTTCTGACCCTCGCCCAAGAAGCA GGTTCGCTGTCCAAGCTCGCCAAACTCTGCGGCTACCGTACCCCCGTCGCACTCTACAAA CTCAAACA? CGCCTTGAAAAGCAGGCAGAAGACCCAGATGCACGCGGCATCCGTCCCAGC CTGATGGC? LAAACTCGAAAAACACACCGGCAAACCCAAAGGCTGGCTCGACAGAARACAC CGCGAACGCACTGTCCCCGAAACCGCCGCAG / AGCACCGGAACTGCCGAAACCCAAATT GCCGAAACCGCATCTGCTGCCGGC7GCCGCAGCGTTACCGTCAACCGCAATACCTGCGAA ACCCAAATCACCGTCTCCATCAACCTCGACGGCAGCGGCAAAAGCAGGCTGGATACCGGC GTACCC TCCTCGAVACA.TGA CGATCAAA CGCCCGCCACGGCATG7v 7GACATCGAC ATCAGCTGCAAAGGCGACCTGCACATC3ACGACCACCACACCGCCGAAGACATCGGCATC ACACTCGGACAAGCAATCCGGCAGGCACTCGGCGACAAAAA GGCATCCGCCGTTACGGA CATTCCTACGTCCCGCTCGACGAAGCCCTC &?????? GCCGCGTCGTCATCGACCTTTCCGGCCGC CCCGGACTCGTGTACAACA7CGAATTTACCCGCGCACTAATCGGACG7TTCGATGTCGAT TTGTTTGARGAATTTTTCCACGGCATCGTCAAC ACAGTATGATGACCCTGCACATCGAC AACCTCAGCGGCAAAAACGCCCACCATCAGGCGGAAACCGTATTCAAAGCCTTCGGGCGC GCCC7GCGTATGGCAGTCGACACGACCCGCGCATGGCAGGACAGACCCCCTCGACCAAA GGCACGCTGACCGCATAA NMB15S3 Protein sequence MNLTKTQRQLHNFLTLAQEAGSLSKLAKLCGYRTPVALY LKQRLSKQAEDPDARGIRPS LMAKLEKHTG PKGWLDRKKRERTVPETAAESTGTAETQIAETASAAGCRSVTVNRNTCE TQITVSINLDGSGKSRLDTGVPFLEHMIDQIARHGMIDIDISCKGDLHIDDHHTAEDIGI TLGQAIRQALGDKKGIRRYGHSYVPLDEALSRVVIDLSGRPGLVYNISFTRALIGRFDVD LFEEFFHGIVNHSKMTLHIDNLSGKNAHHQAETVFKAFGRAIRMAVEHDPRMAGQTPSTK GT TA The following additional antigens were identified using essentially the methodology described above: NMB 1333 Nucleic acid sequence A7GCGCTACAAACCCCTTCTGCTTGCCCTGATGCTCGTTTTTTCCACGCCCGCCG1TGCC GCCCACGACGCGGCACACA? CCGTTCCGCCGA? GTGAAAAMCAGACGAAGAACAAA? AA GAACAGCCC6AAGCGGCGGAAGGCAAAPJIAGAAAAAGGCJ AAAATGGCGCAGTGAAAGAT ? AAAAAa.CAGGCGGCAAAG? CGCGGCAAAAGAGGGCAAAGAGTCCA A? CCGCA? AGAAGCAGAG? AGGAGGCGACATCCAGGC? GTCTGCGCGCAAAGGACGCGAA GGGGATAAGA? ATCGAAGGCGGAACACAAAAAGGCACATGGCAAGCCCGTGTCCGGATCC JJUIGAAAAAAACGC? AAAACACAGCCTG? Í ^ CAAACAA CAGGGCAATCCGCGCAAGGGCGGCAAGGCGGA IAAAGACACTGTTTCTGC / iAA? A? AAAA GTCCG7TCCGACAAGA? ICGGCAAAGCAGTGAAACAGGACAAAA? ATACAGGGAAG? GAAA AATGCCAA? CCGATTCCGACGAATTGAA? GCCGCCGTTGCCGCTGCCACCAATGA-TGTC GAAAACAAAAAAGCCC7GCTCAAACAAAGCGAAGGAATGCTGCTTCATGTCAGCAATTCC CTCA? ACAGCT? CAGG? AGAGCGGATCCGCCAAGAGCG? ATCCGTC? GGCGCGCGGCAAC CTTGCTTCCGTCAACCGCAA? CAGCGCGAGGCTTGGGACAAGTTCCAAAAACTCAATACC GAGCTGAACCGTTTGAAAACGGAAGTCGCCGCTACGAAAGCG GATTTCCCG? 'TTCGTA TCGGSGAACTATAAAAACAGCCAGCCGAATGCGGT ? GCCCTGTTCCTGAAAAACGCCGAA CCGGGTCAGAAAAACCGCTTTTTGCGTTATACGCGTTATGTAAACGCCTCCAATCGGGAA GTTGTCAAGGATT? GGA ^ AAACAGCAGAAGGCTTTGGCGGTACAAGAGCAGAAAATCAAC AAI'GAGCTTGCCCGTTTGAAGAAAATTCAGGCAAACGTGCAATCTCTGCTGAAAAAACAG GGTGTAACCGATGCGGCGGAACAGACGGAAAGCCGCAGACAGAATGCCAAAATCGCC? AA GATGCCCGAAAACTGCTGGAACAGAAAGGGAACGAGCAGCAGCTGAACAAGCTCTTGAGC AJ ^ TTTGG? GAAGAAAAAGGCCGAACACCGCATTCAGGATGCGGAAGCA AAGA? AATTG GCTGAAGCCAGACTGGCGGCAGCCGAAAAAGCCAGAAAAGAAGCGGCCCAGC? GAAGGCT GAAGCACGACGTGCGG? ÍATGTCCAACCTGACCGCCGAAGACAGGAACATCCAAGCGCCT TCGGTTATGGGTATCGGCAGTGCCG? CGGTTTCAGCCGCTGCAAGGACGTTTGAAAAAA CCGGTTGACGGTGTGCCGACCGGACTTTTCGGGCAGAACCGGAGCGGCGGCGATATTTGG AAAGGCGTGTTCTATTCCACTGCACCGGCAACGGTTGAAAGCATTGCGCCGGGAACGGTA AGCTATGCGGACGAGTTGGACGGCTACGGCAAAGTG6TCGTGGTCGATCACGGCGAGAAC TACATCAGCATCTATGCCGGGTTGAGCGAAAGTTCCG? CGGCAAGGGTTATATGGTCGCG GCAGGAAGCAAAATCGGCTCGAGCGGGTCGCTGCCGGACGGGGAAGAGGGGCTTTACCTG CAAATACGTT ?? CAAGGTCAG3TATTGAACCCTTCG? * GCTGGATACGTTGA NMB 1333 amino acid sequence MR YK PLLLALM LV FS T? AV AAH Daah N RS AE VKKQT KKEQ KN P BAAEGKKE KGKNG AVK D KKTGGKE.AAKEGKE5KKTAKNRKEAEKEATSRQSARKGREGDKKSKAEHKKAKGKPVSGS IffiOTAKTQPENKQG SSAKGQGNPRKGGKASKDTVSASKKVRSDK GKAVKQD KYREEK NAKTDSDELKAAVA? ATNDVENKKA1 KQS? GMLLHVSNSLKQLQEERIRQERIRQARGN ASVNRK REA DKFOKLWTELNRLE TEVAATKAQ? SRFVSGNYKNSQPNAVALFLKNAE PGQRTSIRFLRYTRy TaASNREVVKDLE QQKRLAVQEQK? NNELASLKKIQANVQS LKKQ Gv'TDAAEQTBSRRQNAKIAKDARKLL? QKGNEQ LNKLLSNLEKKKAEHRIQDAEAKRKL AEARLAP? EKARKEAAQQKAEARR;? SMSNLTAEDRNIQAPSVMGIGSAl > GFSRtS ?? 5Rl.KK PVDGVPTGLFGQNRSGGDIKKGVFYSTAPATVESIAPGTVSYADELDGYGKVWVDBGEB YISIYAGLSEISVGKGYMVAAGSKIGSSGSLPDGEEGLYLQIRYQGQVLNPSSS? IR NMB 0377 Nucleic acid sequence ATGGCGTTTTGCACCAGTTTGGGAGTGATGATGGAAACACAGCTTTACATCGGCATCATG TCGGGAACCAGCATGGACGGGGCGGATGCCGTACTGATACGGATGGACGGCGGCAAATGG C7GGGCGCGGAAGGGCACGCCTTTACCCCCTACCCCGGCAGGTTACGCCGCCAATTGCTG GATTTGCAGGACACAGGCGCAGACGAACTGCACCGCAGCAGGATTTTGTCGCAAGAACTC A.GCCGCCTATATGCGCAAACCGCCGCCGACTGCTGTGCAGTCAAAACCTCGCACCGTCC GACATTACCGCCCTCGGCTGCCACGGGCAAACCGTCCGACACGCGCCGGAACACGGTTAC AGCATACAGCTTGCCGATTTGCCGCTGCTGGCGGAACGGACGCGGATTTTTACCGGCGGC GAC7TCCGCAGCCGCGACCTTGCGGCCGGCGGACAAGGCGCGCCACTCGTCCCCGCCTTT CACGAAGCCCTGTTCCGCGACAACAGGGAAACACGCGCGGTACTGAACATCGGCGGGATT SCCAACATCAGCGTACTCCCCCCCGACGCACCCGCCTTCGGCTTCGACACAGGGCCGGGC AATATGCTGATGGACGCGTGGACGCAGGCACACTGGCAGCTTCCTTACGACAAAAACGGT GCAA. GGCGGCACAñGGCACATATTGCCGC ^.? IACTGCTCGACAGGCTGCTCGCCCACCCG TATTTCGCACAACCCCACCCTAAAAGCACGG6GCGCGAACTGTTTGCCCTAAATTGGCTC GAAACCTACCTTGACGGCGGCGAAAACCGATACGACGTATTGCGGACGCTTTCCCGTTTT ACCGCGCAAACCGTTTGCGACGCCGTCTCACA.CGCAGCGGCAGATGCCCGTCAA TG GAC ATTTGCGGCGGCGGCATCCGCAATCCTGTTTTAATGGCGGATTTGGCAGAATGTTTCGGC ACACGCGTTTCCCTGCACAGCACCGCCG? CCTGAACCTCGATCCGCAATGGGTGGAAGCC GCCGCA7TTGCGTGGTTGGCGGCGTG7TGGATTA TCGCATTCCCGGTAGTCCGCACAAA GCAACCGGCGCA7CCAAACCG7GT7 \ TTCTGGGCGCGGGATATT? TTAT A NMB0377 amino acid sequence M? FCTSLGVbSMETQ YIGIMSGTSMDGADAVLIRMDGGOTLGAEGHAFTPYPGRLRRQLL DLQDTGADEI.HRSRII.SQEL5RLYAQTAAELLCSOI3LAPSDITALGCHGQTVRHAPEHGy S IQÍiADLPLJiASRTRI FTVGDFRSRDLAAGGQGAPLVPAFHEALFRDNRSTRAVLN I.GGI ANI SVLPPDAPAFGFDTGPGNMUíIiA TQAíiWQLPYDKHGAKAAQGNrLPQL DRLIjAHP YFAC'PHPKSTGRELFALNWLETYLDGGESRYDVLR LSRFTAQTVCDAVSHA ADARQMY ICGGGIRNPVLMADLAECFGTRVSLKSTA LMLDPQ VEAAAFAV? LAAC IiJRI PGSPHK TO? GAS K C I AG AND Y Y NMB0264 Nucleic acid sequence ATGTTGAACAAAATATTTTCCTGGTGCGAGTCCCGAATCGACCCTTATCCCGAAGCCGCC CCGAAAACGCCAGAAAAAGGCTTGTGGCGGTTTGTCTG AGCAGCATGGCCGGCGTGCGG AAATGGATAGCCGCCCTGGCTGCGCTGACCGCCGGCATCGGCATTATGGAAGCCCTGGTT TTTCAATTTATGGGCAAAATCGTGGAGTGGCTCGGCAAATAC6CGCCCGCCGAACTGTTT GCCGAAA? AAGTTGGGAACTGGCGGCAATGGCGGCGATGATGGTATTTTCGGTTGCGTGG GCGTTTGCCGCGTCCAACGTGCGCCTGCAAACCCTTCGGGCGTG? TCCCCATGCGCCTG CGCTGGAACTTCCACCGCCTGATGCTGAACCAAAGCCTCGGTTTTTATCAGGACGAA.TTT GCCGGACGCGTGTCCGCCAAAGTC.ATGCAGACCGCGCTGGCGTTGCGCGACGCGGTGATG? CGGTTGCCGATATGGTCGTTTÁTGTGTCGGTG? ATTTCATTACCTCCGGCGTGATTCTC GCCTCGCTCGACTCATG? CTGCTGCTGCCCTTTATCGGCTGGATTGTCGGTTTCGCTTCG GTGATGCGCCTGCTGATTCCCAAATTGGGGCAAACCGCCGCATGGCAGGCGGATGCCCGC TCGCTGAGGACCGGCCGCATTACCGATGCCTATTCCAATATCGCCACCGTCAAACTCTTC TCCCACGGCGCGCGTGAAGCCGCCTATGCCARGC? GTCGATGGAAGAATT? ATGGTTACG GTGCGCGCCCAAATGCGGCTGGCGACGCTGCTGCATTCGTGCAGCTTCATCGTCAACACC TCCCTGACCCTCTCCACCGCCGCACTGGGCATCTGGCTCTGGCACAACGGGCAGGTCGGC GTGGGCGCGGTTGCTACAGCCACCGCCATGGCGTTGCGCGTCAACGGTTTGTCGCAATAC ATTATGTGGG.AATCCGCGCGGCTGTTTGAAAACATCGGCAACCG? CGGCG? CGGCATGGCA ACCCTGTCCAAA.CCGCACACCATCCTCGACAAGCCCCGGGCACTGCCGCTGAACGTGCCG CAAGGCGCAFITCAAATTTGAACACGTCGATTTCTCCTACGAAGCGGG AAACCGCTGCTC AACGGCTTCAACCTCACCATCCGCCCGGGCGAAAAAGTCGGCTTG? TCGGACGCAGCGGC GCGGGCAAATCCACCATCGTCAACCTGCTTTTGCGCTTCTACGAACCGCAAAGCGGCACG GTTTCGATCGACGGGCAGGACATAAGCGGCGTTACCCAAGAATCTTTACGCGCCCAAATC GGTTTGGTCACGCAAGATACCTCGCTGCTGCACCGTTCCGTGCGCGAC? ACATTATTTAC GGCCGCCCCGACGCGACCGATGCCGAAATGGTT7CTGCCGCCGAACGCGCCGAAGCCGCC GGCTTCATCCCCGACCTTTCCGATGCCAAAGGGCGGCGCGGCTACGACGCACACGTCGGC GAACGCGGCGTGAAACTCTCCGGCGGGCAACGCCAGCGCATCGCCATCGCCCGCGTGATG CTCAAAGACGCACCGATTCTTCTTTTG6ACGAAGCCACCAGCGCGC7CGATTCCGAAGTC GAAGCCGCCATCCAAGA? AGCCTCGACAAAATGATGGACGGCAAAACCGTCATCGCCATC GCCCACCGCCTCTCCACCATCGCCGCAATGGACAGGCTCGTCGTCCTCGACAAAGGCCGC A.TCATCGAAGAAGGCACACACGCCGAACRCCTCGAAÑAACGCGGGCRTTACGCCAAACL'C TGGGCGCACCAGAGCGG CGGCTTCCTCAACGACACGTCGAGTGGCAGCACGACTGA NMB0264 Amino acid sequence HLNKIFS FESRIDPYPEAAPKTPEKGLWRFVHSSMAGVR ííIAAí ALTAGlGIMEALV FQfc'iKSKIVE ^ GKYAPAELFAEKSWElAAAAMMVFSVAWAFAASKVRLQTLQGVFPMRL RWNFHRLMLHQSLGFYQDEFAGRVSAKWfQTAJxAIJlDAVMTVADCr / VYVSVYFITSGVlL ASLDSWLLLPFIGWIVGFASVMRLLIPKLGQTAAKQAD RSLMTGPvITDAYSNIATVKLF SEGARSAAyAKQSMEEFMVTVRAQMRLATLLKSCSFIVNTSLTLSTAALGIWLWHNGQVG VGAVATATAMALRVNGLSQYIMWESARLFENIGTVGDGMATLSKPKTILDKPRALPLNVP GAI FEHVDFSYEAGK LLNGFNLTIRPGEEC \;??? G IGRSGAG STIVWLLLRFYEPQSGT VS DG0DISGVTQF.3LRAQIGLVT0DTSJ..LHRSVRDN IYGRPDATDAEMVSAASRA.SAA GFIPDLSDAKGRRGYDAl'iVGERGVKLSGGQRQRXAIARVI-SLKDAPXLL DSATSALDSEV SAA? ? QESLDKIÍMDGKTVIAIAHR STIAA ^ DRL ^ / DKGRIIEEGTKAEL EKRGLY? KL WAHQSGGF SEHVEWQHD NMB 1036 amino acid sequence MT.AQTLYDKLWNSHVVREEET3GTVLLYIDRIILVHEVTSPQAFEGLKMAGRKLWRIDSVVS 5 ADH NT PTG DWD G IQDPI S KLQVDTLDKN I FG ICE ALAYFPEMDKGQG I VMG VR PEQGAT LPGMTWCGDSKTSTHG? FGALA1ÍGIGTSEVEBTMATQCITAKKSKSMLISVDGKLKAGV T AKD VAL AND 11 GQI GT AGGTG YAI EFGGE AI RS LS MES RMTLCNMAI EAGARS GMVAV ÜQT? ? DY V DKP FAPEGEAWD AVS AND WRTLVS DEGAVFDKE YRFNAED I E PQVT GTS PEMVL DISSKVPNP / ÍEETDPVKRSGMERALEYMGL? AGTPLNEIPVDIVFIGSCTNSRGEDLREA AAIAKDRKKAANVQRYLIVPGSGLVKEQAEKEGLD I FIEAGFEWREPGCSMCLAMNADR LT PGQRCASTSH N FEGRQGN6G THLVS FAMAAAAAVTGRFTDI MMA NMB1 176 Nucleic acid sequence ATGAAAGACAAGCACGATTCTTCCGCCATGCGGCTGGACAAATGGCTTTGGGCGGCACGT TTT77CAAGACCCGTTCCCTTGCGCAAAAGCACATCGAACTGGGTAGGGTTCAAGTAAAC GGCTCGAAGGTCAAAAACAGTAAAACCATAGACATCGGCGATATTATCGACCTGACGCTC AATTCCCTTCCCTATAAAATCAAGGTTAAAGGTTTGAACCACCAACGCCGCCCGGCATCC GAGGCGCGGCTTCTGTATGAAGAGGACGCGAAAACGGCAACATTGAGGGAAGAGCGCAAA CAGCTCGACCAATTCAGCCGCATCACTTCCGCCTATCCCGACGGCAGACCGACCAAGCGC GACCGCCGCCAACTGGACAGGCTGAAAAAAGGAGACTGGTAA NMB1176 Amino Acid Sequence HKDKKDSSWÍRLDK LWAARFFKTRSLAQKKISLGRVQWGSKVKNSKTIDIGDIIDLTL NSLPYKIKVKGLNHQRRPASEARLLYEEDAKTA.TLREERKQLDQFSRITSAYPDGRPTKR DRRQLDRLKKGDW Nucleic acid sequence NMB1359 ATGAACCACACCGTTACCCTGCCCGACCAAACCACCTTTGCCGCCAACGACGGCGAAACC GTTTTGACCGCTGCCGCCCGTCAAAACCTCAACCTGCCCCATTCCTGCAAAAGCGGTGTC TGCGGACAATGCAAAGCCGAACTGGTCAGCGGCGATATTCAAATGGGCGGACACTCGGAA CAGGCTTTATCCGAAGCAGAAAAAGCGCAAGGCAAGATTTTGATGTGCTGCACCACTGCG CAAAGCGATATCAACATCAACATCCCCGGCTACAAAGCCGATGCCCTACCCGTCCGCACC CTGCCCGCACGCATCGAAAGTATTATTTTCAAACACGATGTCGCCCTCCTGÍ? ACTTGCC CTGCCCAAAGCCCCGCCGTTTGCCTTCTACGCCGGGCAATACATTGATTTACTGCTGCCG GGC7iACGTCAGCCGCAGCTACTCCATCGCCAATTTACCCGACCAAGAAGGCATTTTGGAA CTGC? CATCCGCAGGCACGAAAACGGTGTCTGCTCGGAAATGATTTTCGGCAGCGAACCC AAAG7CAAAGAAAAAGGCATCGTCCGCGTTAAAGGCCCGCTCGGTTCGTTTACCTTGCAG GAG? CAGCGGC.AAACCCGTCATCCTGCTGGCAACCGGCACAGGCTACG CCCCATCC6C AGCATCCTGCTCG? CC TATCCGCCAAGGCAGCAACCGCGCCGTCCATTTCTACTGGGGC GCGCGTCATCAGGATGATTTGTATGCCCTCGAAGAAGCACAAGGGTTGGCATGCCGTCTG AAAAACGCCTGCT7CACCCCCGTATTGTCCCGCCCCGGAGAGGGCTGGCAGGGAAGAAAT GGTCACGTACAAGACATCGCGGCACAAGACCACCCCGACCTGTCGGAATACGAAGTArTT CíCCTGCGGTTCTCCGGCCATGACCGAACAAACAAAGAATCTGT TTGTGCAACAGCATAAG CTGCCGGAAAACTTGT TTTCTCC ACGC TTCACGCCGTCCGCATCAT? A NMB 1359 amino acid sequence MNHTVTLPDQTT? AANDGETVLTAAARQNLNLPHSCKSGVCGQCE AEINSGDlQHGGHSE QA SEAEKAQG ILMCCTTAQSDINIHIPGYKADALPVRTLPARIESIIFKHDVALLKLA LPKAPPFAFYAGQYIDLLLPGNVSRSYSIANLPDQEGILELHIRRHENGVCSEMIFGSEP, vrKE GIV ?? RKG? LGSFTLQ? DSGKPVILLATGTGYAi5ÍRS? LLDLJRQGSNRAVHFY5vG ARHQDDLYALEEAQGLACRLKNACFTPVLSRPGEG QGRNGHVQDIAAQDHPDLSSYEVF? CGS PAMT EQTKKLFV QHKLPENLFFSDAFTPSAS NMB 1138 Nucleic acid sequence ATGA? AGACAAGCACGATTCTTCCGCCATGCGGCTGGACAAATGGCTTTGGGCGGCACGT TTTTTCAAGACCCGTTCCCTTGCGCAAAAGCACATCGAACTGGGTAGGGTTCAAGTAAAC GGCTCGAAGGTCAAAAACAGTAAAACCATAG? CATCGGCGATATTATCGACCTGACGCTC AATTCCCTTCCCTATAAAATCAAGGTTAAAG6TTTGAACCACCAACGCCGCCCGGCATCC GAGGCGCGGCTTCTGTATGAAGAGGACGCGAAAACGGCAACATTGAGGGAAGAGCGCARA CAGCTCGACCAATTCAGCCGCATCACTTCCGCCTATCCCGACGGCAa CCGACCAAGCGC G ^? CCGCCGCCAACTGGACAGGCTGAAAAAAGGAGACTGGTAA NMB1138 Amino acid sequence MKDKHDSS? MRLDKWLWAARFFKTRSIAQKHIELGRVQVNGSKVKbISKT? DIGDIIDLTL NSLPYKZKVKGLNHQRRPASEARLLYEEDAKTATLREERKQLDQFSRITSAYPDGRPTKR DRRQLDRLKKGDW Program of SEQ ID Nos SEQ ID No Sequence 1 NMB0341 DNA 2 NMB0341 Protein 3 NMB1583 DNA 4 NMB1583 Protein 5 NMB1345 DNA 6 NMB1345 Protein 7 NMB0738 DNA 8 NMB0738 Protein 9 NMB0792 DNA 10 NMB0792 Protein 1 1 NMB0279 DNA 12 NMB0279 Protein 13 NMB2050 DNA 14 NMB2050 Protein 15 NMB1335 DNA 16 NMB1335 Protein 17 NMB2035 DNA 18 NMB2035 Protein 19 NMB1351 DNA 20 NMB1351 Protein 21 NMB1574 DNA 22 NMB1574 Protein 23 NMB1298 DNA 24 NMB1298 Protein 25 NMB1856 DNA 26 NMB1856 Protein 27 NMB0119 DNA 28 NMB0119 Protein 29 NMB1705 DNA 30 NMB1705 Protein 31 NMB2065 DNA 32 NMB2065 Protein 33 NMB0339 DNA 34 NMB0339 Protein 35 NMB0401 DNA 36 NMB0401 Protein 37 NMB1467 DNA 38 NMB1467 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 NMB0337 DNA 48 NMB0337 Protein 49 NMB0191 DNA 50 NMB0191 Protein 51 NMB1710 DNA 52 NMB1710 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 NMB1036ADN 62 NMB1036 Protein 63 NMB1176ADN 64 NMB1176 Protein 65 NMB1359ADN 66 NMB1359 Protein 67 NMB1138ADN 68 NMB1138 Protein

Claims (6)

NOVELTY OF THE INVENTION CLAIMS

1. - A polypeptide comprising the amino acid sequence selected from any 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 said fragment or variant.

2. A polynucleotide that encodes a polypeptide according to claim 1.

3. A method for preparing a polypeptide according to claim 1, the method comprising the expression of the polynucleotide according to claim 2 in a cell. host and isolating said polypeptide.

4. A method for preparing a polypeptide according to claim 1 comprising chemically synthesizing said polypeptide.

5. The use of a polypeptide as claimed in claim 1 or a polynucleotide according to claim 2 in the manufacture of a vaccine useful for vaccinating an individual against Neisse a meningitidis.

6. - A pharmaceutical composition comprising a polypeptide according to claim 1 or a polynucleotide according to claim 2 and a pharmaceutically acceptable carrier.