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WO2012095684A1 - Procédés de criblage de substances utiles pour prévenir et traiter les infections neisseriennes - Google Patents

Procédés de criblage de substances utiles pour prévenir et traiter les infections neisseriennes Download PDF

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Publication number
WO2012095684A1
WO2012095684A1 PCT/IB2011/000246 IB2011000246W WO2012095684A1 WO 2012095684 A1 WO2012095684 A1 WO 2012095684A1 IB 2011000246 W IB2011000246 W IB 2011000246W WO 2012095684 A1 WO2012095684 A1 WO 2012095684A1
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Prior art keywords
pptb
pilin
neisseria
amino acid
prevention
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English (en)
Inventor
Guillaume DUMENIL
Julia CHAMOT-ROOKE
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Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
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Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
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Priority to PCT/IB2011/000246 priority Critical patent/WO2012095684A1/fr
Publication of WO2012095684A1 publication Critical patent/WO2012095684A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/22—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Neisseriaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1288—Transferases for other substituted phosphate groups (2.7.8)

Definitions

  • the present invention relates to methods for the screening of substances that may be useful for the prevention and treatment of infections by Neisseria bacterial species.
  • Neisserial strains of bacteria are the causative agents for a number of human pathologies, against which there is a need for effective vaccines and therapeutic composition to be developed.
  • Neisseria gonorrhoeae is the etiologic agent of gonorrhea, one of the most frequently reported sexually transmitted diseases in the world with an estimated annual incidence of 62 million cases.
  • the clinical manifestations of gonorrhea include inflammation of the mucus membranes of the urogenital tract, throat or rectum and neonatal eye infections.
  • Ascending gonococcal infections in women can lead to infertility, ectopic pregnancy, chronic pelvic inflammatory disease and tubo-ovarian abscess formation.
  • Septicemia, arthritis, endocarditis and menigitis are associated with complicated gonorrhea.
  • the high number of gonococcal strains with resistance to antibiotics contributes to increased morbidity and complications associated with gonorrhea.
  • Neisseria meningitidis is an important pathogen, particularly in children and young adults. Septicemia and meningitis are the most life-threatening forms of invasive meningococcal disease (IMD). This disease has become a worldwide health problem because of its high morbidity and mortality. Thirteen N. meningitidis serogroups have been identified based on antigenic differences in the capsular polysaccharides, the most common being A, B and C which are responsible for 90% of disease worldwide. Serogroup B is the most common cause of meningococcal disease in Europe, USA and several countries in Latin America. There are diverse problems with the anti-meningococcal vaccines currently available. The vaccines tend indeed to be specific and effective against only a few strains.
  • Neisseria infections represent a considerable health care problem for which no vaccines and therapeutic compositions are available in the case of N. gonorrhoeae or vaccines with limitations on their efficacy and ability to protect against heterologous strains are available in the case of N. meningitidis.
  • the Gram-negative bacterium Neisseria meningitidis is a leading cause of septicemia and meningitis in humans.
  • individual bacteria adhere to the nasopharynx epithelium via their type IV pili, a filamentous organelle common to numerous pathogenic bacterial species.
  • bacteria proliferate on the cellular surface in tight three-dimensional aggregates termed microcolonies.
  • the formation of these aggregates results from homotypic, type IV pili-mediated, contacts between the bacteria themselves and contacts between bacteria and the host cell plasma membrane. Contacts with host cells are enhanced by the formation of bacteria-induced plasma membrane protrusions.
  • N. meningitidis The major component of Neisseria spp. type IV pili (PilE or pilin) is modified with phospho choline (PC), phosphoethanolamine (PE) or phosphoglycerol (PG) (4- 6), however the impact of these unusual posttranslational modifications (PTM) on the pathogenesis of N. meningitides has not yet been investigated.
  • PC phospho choline
  • PE phosphoethanolamine
  • PG phosphoglycerol
  • the present invention relates to a pilin phosphotransferase B (pptB) comprising a polypeptide having an amino acid sequence possessing at least 90% amino acid identity with an amino acid sequence consisting of SEQ ID NO:l or a biologically active fragment thereof.
  • pptB pilin phosphotransferase B
  • the present invention also relates to a method for the screening of substances that may be useful for the prevention and treatment of infections by Neisseria bacterial species comprising a step of determining the ability of a candidate substance to inhibit the activity of a purified pilin phosophotransferase B (pptB) according to the invention.
  • pptB pilin phosophotransferase B
  • the present invention also relates to an inhibitor of a pilin phosphotransferase B according to the invention for use in the prevention and treatment of Neisseria infections in a patient.
  • the present invention also relates to a pharmaceutical composition comprising ca therapeutically effective amount of a pptB inhibitor according to the invention and a pharmaceutically acceptable carrier.
  • type IV pili filamentous organelles referred to as type IV pili, which allow the formation of bacterial aggregates associated with host cells.
  • proliferation of N. meningitidis in contact with host cells increased the transcription of a bacterial gene encoding a transferase that adds phosphoglycerol onto T4P.
  • This unusual posttranslational modification specifically released T4P-dependent contacts between bacteria.
  • this regulated detachment process allowed propagation of the bacterium to new colonization sites and also migration across the epithelium, a prerequisite for dissemination and invasive disease.
  • a first object of the invention consists of a method for the screening of substances that may be useful for the prevention and treatment of infections by Neisseria bacterial species comprising a step of determining the ability of a candidate substance to inhibit the activity of a purified pilin phosophotransferase B (pptB).
  • pptB pilin phosophotransferase B
  • Substances that are inhibitors of pptB may be indeed very useful for limiting or inhibiting propagation of Neisseria bacterial species in the whole organism of a subject exprecially blood stream and brain and moreover for limiting or inhibiting migration of the bacteria across the epithelium.
  • Said substances may represent very efficient tools for prevention of Neisseria infections, and more particularly of the prevention of septicemia and meningitis.
  • said substances may be also useful in therapeutic treatment preferably in combination with antibiotics.
  • purified and “isolated” it is meant, when referring to a polypeptide or a nucleotide sequence, that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type.
  • purified preferably means at least 75% by weight, more preferably at least 85% by weight, more preferably still at least 95 % by weight, and most preferably at least 98% by weight, of biological macromolecules of the same type are present.
  • An "isolated" nucleic acid molecule which encodes a particular polypeptide refers to a nucleic acid molecule which is substantially free of other nucleic acid molecules that do not encode the subject polypeptide; however, the molecule may include some additional bases or moieties which do not deleteriously affect the basic characteristics of the composition.
  • said pptB consists of a polypeptide having an amino acid sequence possessing at least 90% amino acid identity with an amino acid sequence consisting of SEQ ID NO: 1 or a biologically active fragment thereof.
  • a pptB characterized according to the invention, or any biologically active peptide thereof “comprises” a polypeptide as defined above because, in certain embodiments, said pptB may not simply consist of said polypeptide defined above.
  • a pptB characterized according to the invention, or any biologically active peptide thereof may comprise, in addition to a polypeptide as defined above, additional amino acid residues that are located (i) at the N-terminal end, (ii) at the C-terminal end or (iii) both at the N-terminal end and at the C-terminal end of said polypeptide above.
  • a polypeptide or a protein having at least 90% amino acid identity with a reference amino acid sequence possesses at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% amino acid identity with said reference amino acid sequence.
  • the sequences are aligned for optimal comparison purposes. For example, gaps can be introduced in one or both of a first and a second amino acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes.
  • a "biologically active fragment" of a pptB that are defined above, it is intended herein a polypeptide having an amino acid length that is shorter than the amino acid length of the enzyme polypeptide of reference, while preserving the same pptB activity, that is the same specificity of catalytic activity and an activity of at least the same order of magnitude than the activity of the parent enzyme polypeptide.
  • a biologically active fragment of a pptB characterized according to the invention possesses a pptB activity that is assessed, using, as substrates, type IV pilin of Neisseria and phosphoglycerol (PG) (that can be free or incorporated on phospholipids of the bacterium), and then evaluating the capability of said fragment to add said phosphoglycerol on type IV pilin on serines (e.g. serine 69 and/or 93 on sequence SEQ ID NO:l) that is produced.
  • Said fragment consists of a biologically active fragment of a pptB according to the invention if the rate of production of is at least 0.1 the rate of the pptB of SEQ ID NO: 1.
  • a biologically active fragment of a pptB according to the invention has an amino acid length of at least 100 amino acid residues.
  • a biologically active fragment of a pptB according to the invention comprises at least 100 consecutive amino acid residues of a pptB as defined above.
  • a biologically active fragment of a pptB as defined above comprises, or consists of, a polypeptide consisting of 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180
  • said biologically active fragment of a pptB comprises or consists of the periplasmic domain of pptB.
  • said periplasmic domain of pptB corresponds to the last 310 amino acid sequence in SEQ ID NO: 1.
  • a candidate substance to be tested inhibits the catalytic activity of said pptB if the activity of said enzyme, when the candidate substance is present, is lower than when said enzyme is used without the candidate substance under testing.
  • the candidate substances that are positively selected at step d) of the method above are those that cause a decrease of the production rate of the final product by said pptB that leads to less than 0.5 times the production rate of the same enzyme in the absence of the candidate substance, more preferably a decrease that leads to less 0.3, 0.2, 0.1, 0.05 or 0.025 times the production rate of the same enzyme in the absence of the candidate substance.
  • the most active candidate substances that may be positively selected at step d) of the method above may completely block the catalytic activity of said enzyme, which leads to a production rate of the final product by said pptB which is undetectable, i.e. zero, or very close to zero.
  • said enzyme consists of a pptB comprising a polypeptide having an amino acid sequence possessing at least 90% amino acid identity with the amino acid sequence of SEQ ID NO:l, or a biologically active fragment thereof.
  • said enzyme consists of the pptB comprising a polypeptide having the amino acid sequence of SEQ ID NO:l, or a biologically active fragment thereof.
  • said enzyme consists of the pptB of SEQ ID NO: l, or a biologically active fragment thereof.
  • the pptB activity is assessed using, as substrates, type IV pilin of Neisseria and phosphoglycerol ) (that can be free or incorporated on phospholipids of the bacterium).
  • type IV pilin of Neisseria and phosphoglycerol that can be free or incorporated on phospholipids of the bacterium.
  • the one skilled in the art may prepare a reaction mixture comprising (i) purified pptB, (ii) type IV pilin of Neisseria and phosphoglycerol and (iii) optionally the inhibitor candidate substance, in a suitable reaction buffer.
  • the phosphorylation reaction is allowed to proceed during a time period preferably ranging from 1.5 h to 2.5 h, most preferably of about 2 h, at a preferred temperature range between 36.5°C. and 37.5°C, most preferably of about 37°C. Then, when brought to completion, the phosphorylation reaction is stopped, for example by boiling for a time period sufficient to inactivate the pptB, e.g. for a period of time ranging from 3 min to 20 min, most preferably a period of time of about 15 min. Then, the resulting reaction product mixture is centrifuged and an aliquot sample is collected from the supernatant of centrifugation.
  • Said supernatant sample is then used to determine, and usually also quantify, the formation of the pilin modified with phosphoglycerol.
  • the formation of said phosphoform is determined, and usually quantified, by mass-spectrometry as described in Example.
  • the skilled man in the art may also envisage to radioactively label phophoglycerol and then quantified the pilin modified with said radioactively labelled phophoglycerol.
  • any of the pptBs that are defined throughout the present specification can be produced by performing various techniques of protein synthesis that are well known by the one skilled in the art, including chemical synthesis and genetic engineering methods for producing recombinant proteins.
  • any one of the pptBs that are defined throughout the present specification are produced as recombinant proteins.
  • a recombinant form of a pptB of the invention or a biologically active fragment thereof
  • the one skilled in the art may insert the nucleic acid encoding the corresponding polypeptide (SEQ ID NO:2), e.g. into a suitable expression vector and then transform appropriate cells with the resulting recombinant vector.
  • Methods of genetic engineering for producing the polypeptides having a pptB activity according to the invention under the form of recombinant polypeptides are well known from the one skilled in the art.
  • the recombinant vector preferably contains a nucleic acid that enables the vector to replicate in one or more selected host cells.
  • Selection genes will typically contain a selection gene, also termed a selectable marker.
  • Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
  • Expression and cloning vectors usually contain a promoter operably linked to the nucleic acid sequence encoding the polypeptide of interest to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include the ⁇ -lactamase and lactose promoter systems (Chang et al., 1978; Goeddel et al, 1979), alkaline phosphatase, a tryptophan (trp) promoter system (Goeddel, 1980; EP 36,776), and hybrid promoters such as the tac promoter (deBoer et al, 1983). Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding the polypeptide of interest.
  • S.D. Shine-Dalgarno
  • Expression vectors used in eukaryotic host cells will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding polypeptide of interest.
  • a recombinant vector having inserted therein a nucleic acid encoding a polypeptide of interest according to the invention having a pptB activity may be transfected to bacterial cells in view of the recombinant polypeptide production, e.g. E. coli cells as shown in the examples herein.
  • the recombinant polypeptide of interest having a pptB activity may be purified, e.g. by one or more chromatography steps, including chromatography steps selected from the group consisting of affinity chromatography, ion exchange chromatography and size exclusion chromatography.
  • the recombinant polypeptide of interest having a pptB activity may be purified by performing a purification method comprises (a) a step of affinity chromatography, (b) a step of anion exchange chromatography with the eluate of step (a) and (c) a size exclusion chromatography with the eluate of step (b).
  • the purified recombinant polypeptide of interest having a pptB activity may be subjected to a concentration step, e.g. by ultrafiltration, before being stored in an appropriate liquid solution, e;g. at a temperature of -20°C.
  • a recombinant polypeptide of interest having a pptB activity may be produced by known methods of peptide synthesis.
  • the polypeptide sequence of interest, or portions thereof may be produced by direct peptide synthesis using solid-phase techniques. (See, e.g., Stewart et al., 1969; Merrifield, 1963).
  • In vitro protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be accomplished, for instance, with an Applied Biosystems Peptide Synthesizer (Foster City, Calif.) using manufacturer's instructions.
  • Various portions of the polypeptide of interest may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the full-length polypeptide of interest.
  • this invention encompasses methods for the screening of candidate substances that inhibit the activity of a pptB as defined herein.
  • this invention also encompasses methods for the screening of candidate substances, that are based on the ability of said candidate substances to bind to a pptB as defined herein, thus methods for the screening of potentially substances that may be useful for the prevention and treatment of infections by Neisseria bacterial species.
  • the binding assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays, and cell-based assays, which are well characterized in the art.
  • binding assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays, and cell-based assays, which are well characterized in the art.
  • All binding assays for the screening of candidate substances that may be useful for the prevention and treatment of infections by Neisseria bacterial species are common in that they comprise a step of contacting the candidate substance with a pptB as defined herein, under conditions and for a time sufficient to allow these two components to interact.
  • These screening methods also comprise a step of detecting the formation of complexes between said pptB and said candidate substances.
  • screening for substances that may be useful for the prevention and treatment of infections by Neisseria bacterial species includes the use of two partners, through measuring the binding between two partners, respectively a pptB as defined herein and the candidate substance.
  • the interaction is binding and the complex formed between a pptB as defined above and the candidate substance that is tested can be isolated or detected in the reaction mixture.
  • the pptB as defined above or alternatively the anti-Neisseria candidate substance is immobilized on a solid phase, e.g., on a microtiter plate, by covalent or non-covalent attachments.
  • Non-covalent attachment generally is accomplished by coating the solid surface with a solution of the pptB of the invention and drying.
  • an immobilized antibody e.g., a monoclonal antibody, specific for the pptB of the invention to be immobilized can be used to anchor it to a solid surface.
  • the assay is performed by adding the non-immobilized component, which may be labeled by a detectable label, to the immobilized component, e.g., the coated surface containing the anchored component.
  • the non-reacted components are removed, e.g., by washing, and complexes anchored on the solid surface are detected.
  • the detection of label immobilized on the surface indicates that complexing occurred.
  • complexing can be detected, for example, by using a labeled antibody specifically binding the immobilized complex.
  • the binding of the anti-Neisseria candidate substance to a pptB of the invention may be performed through various assays, including traditional approaches, such as, e.g., cross- linking, co-immunoprecipitation, and co-purification through gradients or chromatographic columns.
  • protein-protein interactions can be monitored by using a yeast-based genetic system described by Fields and co-workers (Fields and Song, 1989; Chien et al, 1991) as disclosed by Chevray and Nathans, 1991.
  • Many transcriptional activators, such as yeast GAL4 consist of two physically discrete modular domains, one acting as the DNA- binding domain, the other one functioning as the transcription-activation domain.
  • the yeast expression system described in the foregoing publications (generally referred to as the "two- hybrid system”) takes advantage of this property, and employs two hybrid proteins, one in which the target protein is fused to the DNA-binding domain of GAL4, and another, in which candidate activating proteins are fused to the activation domain.
  • the expression of a GAL1- lacZ reporter gene under control of a GAL4-activated promoter depends on reconstitution of GAL4 activity via protein-protein interaction. Colonies containing interacting polypeptides are detected with a chromogenic substrate for .beta.-galactosidase.
  • MATCHMAKER.TM. A complete kit (MATCHMAKER.TM.) for identifying protein-protein interactions between two specific proteins using the two-hybrid technique is commercially available from Clontech. This system can also be extended to map protein domains involved in specific protein interactions as well as to pinpoint amino acid residues that are crucial for these interactions.
  • Another object of the invention consists of a method for the screening of substances that may be useful for the prevention and treatment of infections by Neisseria bacterial species, wherein said method comprises the steps of:
  • the same method may also be defined as a method for the screening of substances that may be useful for the prevention and treatment of infections by Neisseria bacterial species, wherein said method comprises the steps of:
  • Candidate substances that have been positively selected at the end of any one of the in vitro screening methods of the invention may then tested in various in vitro assays.
  • Said in vitro assays may consist in testing the ability of the positively selected candidate substance to impact aggregation, adhesion to epithelial cells, or transmigration across an epithelial barrier of a Neisseria bacterial species. Typically the assays are described in the Example 1.
  • any substance that has been shown to behave like an inhibitor of a pptB after positive selection at the end of any one of the in vitro screening methods that are disclosed previously in the present specification, may be further in vitro assayed for his impact on aggregation, adhesion to epithelial cells, or transmigration across an epithelial barrier of a Neisseria bacterium.
  • any substance that has been shown to behave like an inhibitor of pptB after positive selection at the end of any one of the screening methods that are disclosed previously in the present specification, may be further assayed for his in vivo activity.
  • any one of the screening methods that are described above may comprise a further step of assaying the positively selected inhibitor substance for its in vivo activity.
  • said further step consists of administering the inhibitor substance to a mammal and then determining the activity of said substance in various assays
  • said assays may consist in inducing a colonization of the throat of an animal with Neisseria meningitidis and evaluated the ability of the inhibitor substance to limit or inhibit the entry of said bacteria in the blood or even brain of the animal.
  • Mammals are preferably non human mammals, at least at the early stages of the assessment of the in vivo antibacterial effect of the inhibitor substance tested. However, at further stages, human volunteers may be administered with said inhibitor substance to confirm safety and pharmaceutical activity data previously obtained from non human mammals.
  • Non human mammals encompass rodents like mice, rats, rabbits, hamsters, guinea pigs.
  • Non human mammals also encompass primates like macaques and baboons.
  • Another object of the present invention consists of a method for the in vivo screening of a candidate substance that may be useful for the prevention and treatment of Neisseria infections which comprises the steps of:
  • step a) performing a method for the in vitro screening of a substance as disclosed in the present specification, with a candidate substance; and b) assaying a candidate substance that has been positively selected at the end of step a) for its in vivo activity.
  • the inhibitor substances selected through any one of the in vitro screening methods above may be formulated under the form of pre- pharmaceutical compositions.
  • the pre-pharmaceutical compositions can include, depending on the formulation desired, pharmaceutically acceptable, usually sterile, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration.
  • the diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological saline, Ringer's solutions, dextrose solution, and Hank's solution.
  • the test composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.
  • compositions comprising such carriers can be formulated by well known conventional methods. These test compositions can be administered to the mammal at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration. The dosage regimen will be determined by taking into account, notably, clinical factors. As is well known in the medical arts, dosages for any one mammal depends upon many factors, including the mammal's size, body surface 3X63.., the particular substance to be administered, sex, time and route of administration and general health.
  • the suitable pre-pharmaceutical compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. If the regimen is a continuous infusion, it should also be in the range of 1 ng to 10 mg units per kilogram of body weight per minute, respectively. Progress can be monitored by periodic assessment.
  • the pre-pharmaceutical compositions of the invention may be administered locally or systemically. Administration will generally be parenterally, e.g., intravenously. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents examples include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and inj ectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, anti-oxidants, chelating agents, and inert gases and the like.
  • the inhibitor substances may be employed in powder or crystalline form, in liquid solution, or in suspension.
  • the injectable pre-pharmaceutical compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain various formulating agents.
  • the active ingredient may be in powder (lyophilized or non-lyophilized) form for reconstitution at the time of delivery with a suitable vehicle, such as sterile water.
  • the carrier is typically comprised of sterile water, saline, or another injectable liquid, e.g., peanut oil for intramuscular injections.
  • various buffering agents, preservatives and the like can be included.
  • Topical applications may be formulated in carriers such as hydrophobic or hydrophilic base formulations to provide ointments, creams, lotions, in aqueous, oleaginous, or alcoholic liquids to form paints or in dry diluents to form powders.
  • carriers such as hydrophobic or hydrophilic base formulations to provide ointments, creams, lotions, in aqueous, oleaginous, or alcoholic liquids to form paints or in dry diluents to form powders.
  • Oral pre-pharmaceutical compositions may take such forms as tablets, capsules, oral suspensions and oral solutions.
  • the oral compositions may utilize carriers such as conventional formulating agents and may include sustained release properties as well as rapid delivery forms.
  • the in vivo activity of the inhibitor substance that is tested blood or tissue samples of the tested animals such as brain samples are collected at determined time periods and bacteria counts are performed, using standard techniques, such as staining fixed slices of the collected tissue samples or plating the collected blood samples and counting the bacterial colonies formed. Then, the values of the bacteria counts found for animals having been administered with increasing amounts of the inhibitor substance tested are compared with the value(s) of bacteria count(s) obtained from animals that have been injected with the same number of bacteria cells but which have not been administered with said inhibitor substance.
  • the candidate substance of may be selected from the group consisting of peptides, petptidomimetics, small organic molecules, antibodies, aptamers or nucleic acids.
  • the candidate substance according to the invention may be selected from a library of substances previously synthesised, or a library of substances for which the structure is determined in a database, or from a library of substances that have been synthesised de novo.
  • the candidate substance may be selected form small organic molecules.
  • small organic molecule refers to a molecule of size comparable to those organic molecules generally sued in pharmaceuticals.
  • the present invention also relates to an inhibitor of pptB for use in the prevention and treatment of Neisseria infections. More particularly, the present invention relates to an inhibitor of pptB for use in the prevention of septicemia and meningitis provoked by Neisseria bacterial species, typically Neisseria meningitis or Neisseria gonorrhoeae.
  • the patient that shall be administered with said inhibitor has been in contact with a person or a group of persons that have been infected by Neisseria bacterial species.
  • the pptB inhibitor is administered concomitantly with antibiotics.
  • a further aspect of the invention relates to a method for treating or preventing a Neisseria infection comprising administering a subject in need thereof with a therapeutically effective amount of a pptB inhibitor according to the invention.
  • a “therapeutically effective amount” is meant a sufficient amount of the pptB inhibitor to treat or prevent Neisseria infection at a reasonable benefit/risk ratio applicable to any medical treatment.
  • the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts.
  • the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
  • the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the agent for the symptomatic adjustment of the dosage to the subject to be treated.
  • a medicament typically contains from about 0.01 mg to about 500 mg of the agent, preferably from 1 mg to about 100 mg of the agent.
  • An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
  • a further aspect of the invention relates to method for preventing septicemia and meningitis provoked by Neisseria comprising comprising administering a subject in need thereof with a therapeutically effective amount of a pptB inhibitor according to the invention.
  • the method comprises administering the pptB inhibitor to a surface which has come in contact with or could come in contact with the organism.
  • the method which comprises administering the pptB inhibitor to a mucous membrane of a human subject, may be used to prevent or reduce the symptoms of gonnococcal or meningococcal disease in the human subject.
  • the pptB inhibitor may be incorporated into a pharmaceutical composition which is applied to the mucous membrane of a carrier of the bacterium or a person who could come into contact with the carrier.
  • Pharmaceutical compositions used in the present methods comprise a therapeutically effective amount of a pptB inhibitor and a pharmaceutically acceptable carrier.
  • the composition comprises a relatively inert carrier.
  • compositions may also contain diluents, fillers, salts, buffers, stabilizers, solubilizers, and other pharmaceutically acceptable materials well known in the art.
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the anti-microbial activity of the pptB inhibitor.
  • a pharmaceutical composition comprising a therapeutically effective amount of the pptB inhibitor is applied to a potential or actual site of infection in the host subject before or after the host subject is exposed to the bacterium.
  • composition may be used prophylactically to prevent or reduce the severity of infections of the eye, nose, mouth, throat, oropharynx, genitalia, and rectum.
  • dentrifices, mouthwashes, tooth paste or gels, or mouth sprays are used.
  • Vaginal or rectal administration may be by the usual carriers such as douches, foams, creams, ointments, jellies, and suppositories, the longer lasting forms being preferred.
  • Ocular administration is preferably by ophthalmic ointments or solutions.
  • the pharmaceutical composition may further contain other agents which either enhance the activity of the pptB inhibitor or complement its activity or use in inhibiting growth of the gonoccocus or meningococcus. Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with the pptB inhibitor, or to minimize side effects.
  • the pharmaceutical composition comprises a solvent for the pptB inhibitor such as, for example, an alcohol.
  • a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, corn oil, or synthetic oils may be added.
  • the liquid form of the pharmaceutical composition may further contain a physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol.
  • the preparation o f such pharmaceutical composition having suitable pH, isotonicity, and stability, is within the skill in the art.
  • Administration of the pharmaceutical composition to an uninfected subject is via local administration to a site which has been or may be contacted with the pathogenic organism. It is preferred that the pharmaceutical composition be applied prior to exposure to the targeted pathogen or preferably within 1-24 hours, more preferably within 1-12 hours after exposure of the uninfected subject to the pathogenic organsim.
  • Administration of the pharmaceutical composition to a carrier of Neisseria meningiditis is via local administration to the upper respiratory tract, i.e. ororpharynx.
  • Administration of the pharmaceutical composition to a carrier of Neisseria gonorrhea is via local administration to the genitalia, rectum, or oropharynx.
  • a pilin phosphotransferase B comprising a polypeptide having an amino acid sequence possessing at least 90% amino acid identity with an amino acid sequence consisting of SEQ ID NO:l or a biologically active fragment thereof.
  • the invention relates to a pilin phosphotransferase B (pptB) comprising a polypeptide having the amino acid sequence of SEQ ID NO:l, or a biologically active fragment thereof.
  • the invention relates to a pilin phosphotransferase B (pptB) consisting of SEQ ID NO:l or a biologically active fragment thereof.
  • said biologically active fragment thereof consists of the periplasmic domain of pptB.
  • said biologically active fragment consists of a polypeptide having an amino acid sequence possessing at least 90% amino acid identity with an amino acid sequence consisting of the last 310 amino acid of SEQ ID NO: 1.
  • a still further object of the present invention relates to a nucleic acid that encodes a pilin phosphotransferase B (pptB) according to the invention or a biologically active fragment thereof.
  • said nucleic acid consists of nucleic acid sequence possessing at least 90% amino acid identity with a nucleic acid sequence consisting of SEQ ID NO:2.
  • said nucleic acid is SEQ ID NO:2.
  • Both polypeptides or nucleic acids of the invention are preferably under a purified form.
  • Nucleic acids of the invention may be inserted into suitable vectors, particularly expression vectors, such as those that are described elsewhere in the present specification.
  • Recombinant vectors comprising a nucleic acid as defined above that is inserted therein are also part of the invention.
  • Host cells particularly prokaryotic cells including yeast cells and cells from E. coli that have been transfected or transformed by a nucleic acid above or a recombinant vector above form also part of the present invention.
  • Such recombinant host cells are for example those that are described elsewhere in the present specification.
  • Polypeptides of the invention are preferably recombinantly produced, illustratively according to any one of the techniques of production of recombinant proteins that are disclosed elsewhere in the present specification.
  • a yet further object of the present invention consists of an antibody directed against pilin phosphotransferase B (pptB) that is disclosed in the present specification, or to a biologically active peptide fragment thereof Any one of these antibodies may be useful for purifying or detecting the corresponding pilin phosphotransferase B (pptB).
  • pptB pilin phosphotransferase B
  • antibodies encompassed by the present invention there is no particular limitation on the antibodies encompassed by the present invention, as long as they can bind specifically to the desired pilin phosphotransferase B (pptB) or the desired biologically active fragment thereof. It is possible to use mouse antibodies, rat antibodies, rabbit antibodies, sheep antibodies, chimeric antibodies, humanized antibodies, human antibodies and the like, as appropriate. Such antibodies may be polyclonal or monoclonal, but are preferably monoclonal because uniform antibody molecules can be produced stably. Polyclonal and monoclonal antibodies can be prepared in a manner well known to those skilled in the art.
  • monoclonal antibody-producing hybridomas can be prepared using known techniques, as follows. Namely, the desired antigen or the desired antigen-expressing cell is used as a sensitizing antigen and immunized in accordance with conventional procedures for immunization. The resulting immunocytes are then fused with known parent cells using conventional procedures for cell fusion, followed by selection of monoclonal antibody-producing cells (hybridomas) through conventional screening procedures. Preparation of hybridomas may be accomplished according to, for example, the method of Milstein et al. (Kohler, G. and Milstein, C, Methods Enzymol. (1981) 73:3-46). If an antigen used is less immunogenic, such an antigen may be conjugated with an immunogenic macromolecule (e.g., albumin) before use in immunization.
  • an immunogenic macromolecule e.g., albumin
  • antibody genes are cloned from hybridomas, integrated into appropriate vectors, and then transformed into hosts to produce antibody molecules using gene recombination technology.
  • the genetically recombinant antibodies thus produced may also be used in the present invention (see, e.g., Carl, A. . Borrebaeck, James, W. Larrick, «Therapeutic monoclonal antibodies», Published in the United Kingdom by MacMillan Publishers Ltd, 1990). More specifically, cDNA of antibody variable domains (V domains) is synthesized from hybridoma mRNA using reverse transcriptase. Upon obtaining DNA encoding the target antibody V domains, the DNA is ligated to DNA encoding desired antibody constant domains (C domains) and integrated into an expression vector.
  • V domains antibody variable domains
  • the DNA encoding the antibody V domains may be integrated into an expression vector carrying the DNA of the antibody C domains.
  • the DNA construct is integrated into an expression vector such that it is expressed under control of an expression regulatory region, e.g., an enhancer or a promoter.
  • Host cells are then transformed with this expression vector for antibody expression.
  • an expression regulatory region e.g., an enhancer or a promoter.
  • Host cells are then transformed with this expression vector for antibody expression.
  • any suitable combination of host and expression vector can be used for this purpose.
  • animal cells, plant cells and fungal cells may be used.
  • Animal cells known for this purpose include (1) mammalian cells such as CHO, COS, myeloma, BHK (baby hamster kidney), HeLa and Vero, (2) amphibian cells such as Xenopus oocytes, and (3) insect cells such as sf9, sf21 and Tn5.
  • Plant cells include those derived from Nicotiana plants (e.g., Nicotiana tabacum), which may be subjected to callus culture.
  • Fungal cells include yeasts such as Saccharomyces (e.g., Saccharomyces serevisiae) and filamentous fungi such as Aspergillus (e.g., Aspergillus niger).
  • Bacterial cells known for this purpose are E. coli and Bacillus subtilis.
  • Antibodies can be obtained by introducing target antibody genes into these cells via transformation and then culturing the transformed cells in vitro.
  • the present invention also relates to compositions or kits for the screening of substances that may be useful for the prevention and treatment of infections by Neisseria bacterial species.
  • compositions or kits comprise a purified pilin phosphotransferase B (pptB) preferably under the form of a recombinant protein.
  • pptB pilin phosphotransferase B
  • said pilin phosphotransferase B may be under a solid form or in a liquid form.
  • Solid forms encompass powder of said pilin phosphotransferase B (pptB) under a lyophilized form.
  • Liquid forms encompass standard liquid solutions known in the art to be suitable for protein long time storage.
  • said pilin phosphotransferase B is contained in a container such as a bottle, e.g. a plastic or a glass container.
  • kits may comprise also one or more reagents, typically one or more substrate(s), necessary for assessing the enzyme activity of said pilin phosphotransferase B (pptB).
  • a kit according to the invention comprises one or more of each of the containers described above.
  • the invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.
  • EXAMPLE 1 MATERIALS AND METHODS
  • N. meningitidis strains described in this study were derived from the recently sequenced 8013 serogroup C strain [http://www.genoscope.cns.fr/agc/nemesys] (C. Rusniok et al., Genome Biol 10, Rl lO (Oct 9, 2009).).
  • N. meningitidis strains were grown on GCB agar plates (Difco) containing Kellogg's supplements, in a moist atmosphere containing 5% C02 at 37°C. GFP was expressed by introducing the pAM239 plasmid by conjugation (E.
  • MS/MS experiments were performed using a variable collision energy (10-30 eV), which was optimized for each precursor ion.
  • Mass Lynx 4.1 was used both for acquisition and data processing.
  • Deconvolution of multiply charged ions into neutral species was performed using MaxEntl in the mass range [10 - 25 kDa] with a resolution of 0.01 Da/channel.
  • An external calibration in MS was done with clusters of phosphoric acid (0.01M in 50:50 Acetonitrile:H20 v:v). The mass range for the calibration was m/z 70 - 2000.
  • HEC-IB human endometrial cell line
  • Caco-2 human intestinal epithelial cell line
  • MOI 106 bacteria
  • infected cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) lh at room temperature. Samples were washed three times for 5 min in 0.2 M cacodylate buffer (pH 7.2), fixed for 1 h in 1% (wt/vol) osmium tetroxide in 0.2 M cacodylate buffer (pH 7.2), and then rinsed with distilled water. Samples were dehydrated through a graded series of 25, 50, 75 and 95% ethanol solution (5 min each step). Samples were then dehydrated for 10 min in 100% ethanol followed by critical point drying with C02.
  • N. meningitidis pilin and pilus structure Due to the high sequence identity (77 %) the sequences of N. gonorrhoeae and N. meningitidis pilins could be simply aligned by eye.
  • the basis of the modeling was the N. gonorrhoeae pilin structure as deposited in the model of the pilus (PDB code 2HIL). Missing backbone and side chains were added and optimized for packing within the context of the pilus, in a multi-stage procedure that we implemented in the program CNS (A. T. Brunger et al., Acta Crystallogr D Biol Crystallogr 54, 905 (Sep 1, 1998).).
  • the first stage is a quick optimization of the geometry and the packing, with a simplified non-bonded interaction (repulsive Van der Waals only). During this stage, positional restraints were used on those residues that were strictly identical to the residues in the N. meningitidis pilin.
  • the second stage is a refinement in vacuo, using adapted non- bonded parameters (a distance-dependent dielectric, a switching function between 2 and 9 A, and a non-bonded cut-off of 10 A).
  • the third stage is a short refinement in water, similar to the one used in NMR structure determination (J. P. Linge, M. A. Williams, C. A. Spronk, A. M. Bonvin, M.
  • the CHARMM19 force field was extended for the serine modifications (B. R. Brooks et al, J Comp Chem 4, 187 (1983).). Topology and parameter files for these modifications were obtained with the help of the PRODRG2 server (A. W. Schuttelkopf, D. M. van Aalten, Acta Crystallogr D Biol Crystallogr 60, 1355 (Aug, 2004).). The atom types were as far as possible mapped onto those of the CHARMM19 force field, or, if not possible, onto those of the CHARMMl 1 force field (for example, for the glycerophosphate group).
  • Bundles of pili were generated as symmetric antiparallel tetramers by randomly varying the distance, the rotation angle around the long axis of a pilus, and the crossing angle between pili.
  • the energetic analysis was performed with the ACE generalized Born model implemented in CNS for symmetric systems (L. Moulinier, D. A. Case, T. Simonson, Acta Crystallogr D Biol Crystallogr 59, 2094 (Dec, 2003).).
  • the binding energy was estimated as the difference between the electrostatic, van der Waals and generalized Born contributions to the total energy calculated in the complex and in an isolated pilus. We used 6 for the internal dielectric and 80 for the external dielectric.
  • 2D gel electrophoresis The isoelectric point of the major pilin subunit in different conditions was determined by 2D gel electrophoresis followed by immunoblot and detection of PilE with specific antiserum. Infection of an epithelial monolayer growing in a 6-well plate was initiated for a period of 30 min at an MOI of 400, cells were washed, infection was allowed to proceed for 2-4 hours as indicated, rinsed with PBS and loading buffer added directly in the wells (8 M urea, 2 M thiourea, 4% (w/v) CHAPS). All samples were treated with 2D Clean-Up kit (GE Healthcare) according to the manufacturer's instructions and the resultant dry pellets were resuspended in loading buffer.
  • 2D Clean-Up kit GE Healthcare
  • Neisseria meningitidis is a leading cause of septicemia and meningitis in humans (1). Initially, individual bacteria adhere to the nasopharynx epithelium via their type IV pili, a filamentous organelle common to numerous pathogenic bacterial species (2). In the following hours, bacteria proliferate on the cellular surface in tight three-dimensional aggregates termed microcolonies. The formation of these aggregates results from homotypic, type IV pili-mediated, contacts between the bacteria themselves and contacts between bacteria and the host cell plasma membrane. Contacts with host cells are enhanced by the formation of bacteria-induced plasma membrane protrusions (3).
  • N. meningitidis The major component of Neisseria spp. type IV pili (PilE or pilin) is modified with phospho choline (PC), phosphoethanolamine (PE) or phosphoglycerol (PG) (4-6).
  • PC phospho choline
  • PE phosphoethanolamine
  • PG phosphoglycerol
  • a whole protein mass spectrometry approach was chosen to determine the phosphorylation state of type IV pili (7, 8).
  • Analysis of purified pili from the well- characterized 8013 strain (9) grown on solid medium yielded a main peak with a mass of 17491 Da and a minor secondary peak with a mass of 17645 Da corresponding to the addition of one phosphoglycerol (154 Da).
  • Analysis of purified pili from strains carrying point mutations substituting conserved serine residues 69 and 93 of the PilE protein into an alanine showed that all pilin subunits were modified with PG on serine 69 (17491 Da) while only about 15% of pilin subunits were also modified on serine 93 (17645 Da).
  • the NMV 0885 gene (ortholog of NMA1705 and NMB1508) was a good candidate to carry out this activity because it is part of the cluster of orthologous group entitled "Phosphoglycerol transferase and related proteins" (COG1368, (10)).
  • Analysis of type IV pili purified from a strain carrying a deletion in NMV_0885 gene revealed a single peak of 17337 Da corresponding to pilin without any PG demonstrating that this gene is responsible for the transfer of PG onto the pilin.
  • transferase pptB platinumin phosphotransferase B
  • the pptB gene was previously described as a member of a group of 16 N. meningitidis genes containing a two-component system regulated promoter referred to as CREN for "Contact Regulatory Element of Neisseria" (11-13). Transcription of pptB increased 2 to 3- fold over a period of 4 hours after adhesion to epithelial cells (11) suggesting that modification of type IV pili with PG could be triggered upon contact with host cells. To test this possibility we expressed the pptB gene under the transcriptional control of the IPTG inducible lac promoter to mimic the 3-fold induction found on cells. In the presence of inducer, the peak corresponding to 2 PG modifications (17645 Da) became the most abundant form.
  • Type IV pili bundle formation and N. meningitidis aggregation are linked (15). Deletion of the pptB gene or substitution of serine 93 with an alanine led to increased aggregate formation in suspension and consistently increased transcription of the pptB gene abrogated bacterial aggregation. The effect of increased pptB transcription on aggregation was rescued by the S93A point mutation. Increased modification of serine 93 with PG thus strongly reduces type IV pili dependent bacterial aggregation by introducing a negative charge at this site. This anti-aggregative effect appeared to overcome the pro-aggregative activity of the minor pilin PilX.
  • the effect of pilin modification with PG on adhesion to epithelial cells was evaluated.
  • the first step of adhesion which is the contact of individual bacteria with the cell surface (Fig. 4A), occurred independently of the level of glycerophosphorylation.
  • Bacterial microcolonies formed by the strains affected in pilin modification with PG did not appear morphologically different from the wild type multilayered microcolonies.
  • pilin glycerophosphorylation had little effect on the total number of cell associated bacteria after 6 hours (Fig. 4D).
  • the number of bacteria disengaging from microcolonies over time was determined.
  • a laminar flow chamber was used as a tool to progressively collect detaching bacteria (Fig. 4E, Fig. S7A). Whereas the number of wild-type bacteria released from the infected monolayer slowly increased with time after 3 hours of infection, detachment of the ⁇ mutant was significantly impaired (1.5x106 vs. 3.4x105 bacteria per ml at 7 hours). About 20-30% of bacteria adhering at 6 hours detached in the following hours of infection. The increase in pilin glycerophosphorylation thus favors the release of a proportion of individual bacteria from the microcolonies that bears little impact on the number of adhering bacteria.
  • Neisseria meningitidis adopts a "multiply and run” strategy, presumably selected and fine-tuned through evolution as a propagation mechanism, key for survival of the bacterium in nature. Because selection of this property in the context of the commensal lifestyle of the bacteria also favors transmigration across the epithelium it is likely to impact human health by favoring invasive infections.

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Abstract

Cette invention concerne une piline phosphotransférase B (pptB) comprenant un polypeptide ayant une séquence d'acides aminés présentant une identité d'au moins 90 % avec une séquence d'acides aminés constituée de SEQ ID No: 1 ou d'un fragment biologiquement actif de celle-ci. Cette invention concerne également un procédé de criblage de substances qui peuvent être utiles pour prévenir ou traiter les infections par l'espèce bactérienne Neisseria comprenant une étape consistant à déterminer l'aptitude d'une substance candidate à inhiber l'activité d'une piline phosphotransférase B (pptB) purifiée selon l'invention. Cette invention concerne également un inhibiteur de piline phosphotransférase B selon l'invention destiné à être utilisé pour prévenir et traiter les infections neisseriennes chez un patient. Cette invention concerne également une composition pharmaceutique contenant une quantité thérapeutiquement efficace d'un inhibiteur de pptB selon l'invention et un véhicule de qualité pharmaceutique.
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Citations (2)

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