Classifications

• • 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
  • C07K14/23—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Brucella (G)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to genetically modified Brucella cells including one or more modifications genetic in wild nucleotide sequences encoding the flagellar proteins of Brucella important in the infectious cycle of this organism in mammals.
• the present invention also relates to a pharmaceutical composition comprising these genetically modified Brucella cells and a method for obtaining such cells and such a pharmaceutical composition.
• Brucella and brucellosis The bacteria of the genus Brucella are gram-negative coccobacilli which belong to the family of ⁇ -Proteobacteria. These are intracellular pathogens that can be cultivated in the laboratory, but of which no natural free form is known. Different species have been defined according to their propensity to be associated with a particular host (for review (Moreno, (2002)). We distinguish B. meli tensis in goats, B. abortus in cattle, B. canis in dog, B. suis in pigs, B. ovis in sheep and B. neotomae in some rodents. Two new species associated with marine mammals are being described, B.
• brucellosis is a disease of the reproductive system inducing abortions in pregnant females and orchitis in males. Transmission from one animal to another seems mainly due to abortions which release a high number of bacteria into the environment. Transmission to humans occurs either through direct contact with infected runts, or through consumption of unpasteurized dairy products. In humans, only the meli tensis, suis, abortus and canis species are known to induce a systemic infection which can take various forms (meningitis, endocarditis, arthritis, etc.) and often associated with undulating fever, called Malta fever. .
• Brucella infects its host are still poorly understood. It could passively penetrate via ruptures of the integrity of the epithelium, but also by crossing the respiratory and / or digestive mucous membranes. Once in the underlying tissue, the bacteria will face the host's immune system. To guard against this, Brucella has developed an original strategy which consists in "hiding" within it. The bacterium uses phagocytic cells as the first replication niche. The immune cells will therefore become Trojans allowing the spread of the bacteria throughout the body. In animals, Brucella is most commonly associated with lymphoid tissue, the mammary gland, and the reproductive organs.
• the pathogenesis induced by Brucella is therefore mainly based on its ability to replicate in eukaryotic cells (phagocytic and non-phagocytic).
• the molecular mechanisms involved in these processes have been the subject of numerous studies. These, using various approaches (deletion, transpositional mutagenesis, DFI, STM ”) in various infection models (macrophages of different species, HeLa cells, BALB / C and C57 Black mice, goats ...) have enabled the identification of more than 200 virulence genes. Analysis of these makes it possible to infer the crucial stages of the Brucella infectious cycle.
• Comp Genomics. 3 (1): 21-9) discovered 3 CDS homologous to flagellar genes in the B genome. abortus.
• the present invention aims to characterize genetic sequences specific for Brucella, the genetic modification makes it possible to select recombinant Brucella cells having a lower infectious power (attenuated or absent virulence) and which can be used as a therapeutic alternative, in particular a vaccine alternative to the vaccine compositions of the prior art.
• the present invention also aims to provide a method for obtaining said recombinant Brucella cells and said pharmaceutical composition of the invention.
• the present invention relates to recombinant Brucella cells comprising a genetic modification in a wild nucleotide sequence encoding a flagellar protein or a nucleotide sequence regulating the wild nucleotide sequence encoding said flagellar protein.
• the Brucella cells are chosen from the group consisting of the following species: Brucella eli tensis (particularly active in goats Brucella abortus, in cattle; Brucella canis, in dogs; - Brucella suis, in pigs; Brucella ovis, in sheep; Brucella neotomae, active in certain rodents) Brucella cetaceae, and Brucella pinnipediae.
• Brucella eli tensis particularly active in goats Brucella abortus, in cattle
• Brucella canis in dogs
• Brucella suis in pigs
• Brucella ovis in sheep
• Brucella neotomae active in certain rodents
• Brucella cetaceae and Brucella pinnipediae.
• This wild-type sequence is preferably selected from the group consisting of the sequences SEQID No. 1 to SEQID No. 50.
• said nucleotide sequences have been modified by an insertion, a deletion or a substitution of at least one nucleotide in the wild-type nucleotide sequence, so as to lose the infectious (virulent) character of the Brucella cells.
• Another aspect of the present invention relates to a vaccine composition comprising a suitable pharmaceutical carrier or diluent, the recombinant Brucella cells of the invention and optionally one or more adjuvants to induce an immune response against a Brucella strain in a mammal, including humans.
• Another aspect of the present invention relates to the use of the composition according to the invention, for the preparation of a medicament intended for the treatment or the prevention of infections induced by Brucella in an animal, in particular goats , cattle, dogs, pigs, sheep (especially sheep) and possibly humans.
• infections are likely to cause abortions in pregnant females and orchitis in males.
• Infections in mammals are also capable of infecting humans and causing a systematic infection which can take various forms, such as meningitis, endocarditis, arthritis and often associated with undulating fever, called Malta fever.
• the present invention also relates to the method of treatment and / or prevention of an infection induced in a mammal, including man, comprising the administration of a sufficient amount of the pharmaceutical composition of the invention for prevent or treat the symptoms of this infection.
• a last aspect of the present invention relates to a process for the preparation of a recombinant Brucella cell which consists in:
• a “virulent” nucleotide sequence encoding a Brucella flagellar protein that is to say a nucleotide sequence capable of being involved in the infectious cycle of this microorganism in a mammal (including humans)
• this nucleotide sequence being identified in the genome of a Brucella strain by the use of a nucleotide sequence specific for a nucleotide sequence motif encoding a flagellar protein, for example motifs characteristic of the sequences SEQID n ° 1 to SEQID No.
• the recombinant Brucella strains collect the recombinant Brucella strains (genetically modified), having this genetic modification (for example, by insertion of a genetic sequence, by deletion of a genetic sequence or by modification of at least one nucleotide and possibly incorporate this cell of Genetically modified brucella comprising this avirulent nucleotide sequence encoding a flagellar protein in a pharmaceutical composition, preferably a vaccine composition, comprising one or more pharmaceutical carriers or diluents suitable for administration to a mammal.
• the genetic modifications were introduced into nucleotide sequences which, unexpectedly, encode flagellar proteins of Brucella which are important in the infectious cycle of this microorganism in mammals.
• sequences were chosen because they represent all of the proteins necessary for the biosynthesis of the secretory apparatus of flagellar proteins to the ultra flagellar structure proper and to the motor as well as proteins involved in the regulation of the expression of these proteins.
• no coding sequence related to the chemotactic system was identified among these sequences.
• these sequences are involved in the infectious cycles in mice (in particular the sequences fliC, flgE, motB, flgl, flhA, 2C2 and fliF identified respectively by SEQID N ° 1, 10, 5, 40, 17 , 18, 9 and 2.
• the present invention also encompasses the genetic modifications of the portions and variants of these sequences or of their complementary strands
• Sections of sequences according to the invention relate to nucleotide or peptide sequences having the same characteristics antigenic and immunogenic only the complete portion, as well as portions also capable of causing infectious cycles in mammals, in particular in the mouse model.
• Variants of these sequences include sequences having a strong homology or a strong identity of sequences with the sequences of the invention [0021]
• this identity or sequence homology is greater than 80 %, preferably greater than 85%, more particularly greater than 95%, or even greater than 98 or 99% with the nucleotide sequence or complete peptide chosen from the group consisting of the sequences SEQID No.
• the variants of the invention may be natural variants (possibly present in ⁇ other families of strains or in other species) or variants modified by mutagenesis techniques or by direct synthesis, but retaining the biological activity of the reference polypeptide, in particular these antigenic and immunogenic properties.
• the antigenic properties can be tested by standard procedures (immunobloting) preferably using polyclonal or monoclonal sera, in particular in standard ELISA tests.
• sequence identity or “sequence homology” are terms well known to those skilled in the art (Carillo, H., and Lipton, D., SIAM J Applied Math 1998, Vol. 48 , p. 1073). Sequence identity can be calculated by those skilled in the art using well known techniques (Computational molecular biology, Lesk, A.MK, ed. Oxford University Press, New York 1988; Biocomputing: informatics and genome projects, Smith , DW, éd.
• portions or fragments of the polypeptide and nucleotide sequences of the invention comprise important structural and functional sequences, in particular the sequences comprising alpha helices or beta planes, the loop-forming regions, the hydrophilic regions or the hydrophobic regions. and the portions comprising high epitope or linear or conformational antigenic indices.
• FIG. 1 represents the monitoring of the expression of the fliF promoter by observation of the GFp fluorescence.
• Figure 2 shows the analysis of the attenuation of flagellar mutants in the mouse model.
• Figure 3 shows the expression of genes and flagellar proteins of -B-rucella depending on the growth phase.
• Figure 4 shows the electron microscopic observation of the Brucella flagellum.
• Figure 5 shows the measurement of the activity of the FliF promoter during a growth curve.
• Figure 6 shows the CDS page analysis and western blotting of the expression of the elbow protein of the Brucella flagellum.
• Figure 7 shows the expression of the elbow protein of the Brucella flagellum.
• FIG. 8 represents the follow-up in ELISA of serology in 5 ewes injected with the mutant Flgl (F) and of the vaccine strain rev1 (R). Detailed description of the invention Characterization of the flagellar sequences
• the inventors have demonstrated the expression of three sequences of the invention: fliF (BMEII0151) ex vivo and fliF (BME --- I0151), flgE (BMEII0159) and fliC ( BMEII0150) in vitro.
• the expression of these three sequences which unexpectedly represent the different levels of the flagellar structure (the monomers of the MS ring, the elbow and the filament) was coordinated.
• These various data make it possible to propose the existence of a functional flagellar regulon in Brucella.
• the Brucella flagellar system is related to that of other rhizobiacs. Within this group, Brucella's closest relative is
• flagellar CDS The technical difficulties linked to experimental infections have led the inventors to seek a condition for in vitro expression of flagellar CDS.
• the expression of these CDS is concerted ( Figure 3).
• Figure 3 The galactosidase activity of B. eli tensis 16M carrying a pfliF-lacZ translational fusion cultivated in a rich medium, shows that the fliF promoter is transiently expressed during the very start of the exponential phase
• Brucella and others establish a scheme for the regulation of this system, a scheme which can itself be integrated with that of other systems involved in virulence.
• Yersinia enteroli tica a phospholipase and probably other proteins involved in virulence are secreted by the apparatus for the export of flagellar proteins.
• Brucella may secrete proteins involved in virulence. It is difficult to understand the interest for the bacterium to synthesize a filament knowing the important metabolic cost for the cell.
• a secretory function would be an accessory function which the flagellar CDS could fulfill.
• Other functions can be envisaged such as adhesion or else immunomodulatory properties.
• Different criteria can be used for select the potentially secreted effectors: have a kinship with a eukaryotic CDS, be next to a small discomfort that can serve as a chaperone, not have a signal sequence.
• a list of 72 CDS presenting homologies mainly with eukaryotic CDS was established. Most of these CDS are homologous to metabolic CDS and one can hardly predict a role for them in the infectious cycle, it is difficult to select them for later experiments.
• Protein extracts can be produced on culture supernatants of a wild Brucella and of a strain disrupted for the secretory apparatus (the mutant flif or flhA for example) and a proteomic approach will allow the comparison of the two strains in order to identify these potential candidates.
• 2C2 encodes an activator of the transcription of the fliF operon.
• the expression of 2C2 (ftcR) is shown in Figures 5 and 6.
• FIG. 5 represents the activity of the fliF promoter during a growth curve was compared (by assaying galactosidase activity) in a WT strain and in various mutants of transcriptional regulators suspected of being involved in regulation of the Brucella flagellar system.
• the activity of pfliF is virtually zero in the 2C2 mutant (also called ftcR for flagellar two component regulator) and the Vjbr mutant (Vacuolar jacking brucella regulator).
• the tetR mutant gives a reduction in activity.
• the activity of the fliF promoter during a growth curve was compared (by assaying galactosidase activity) in a WT strain and in various mutants of transcriptional regulators suspected of being involved in the regulation of the flagellar system of Brucella.
• the activity of pfliF is virtually zero in the 2C2 mutant (ftcR).
• Brucella samples taken at the start of the exponential phase (4, 8 and 11 h of culture) were analyzed by SDS PAGE and western blotting with anti-elbow monomer antibodies (FlgE).
• the regulator is generally in operon with its histidine kinase.
• 2C2 is not located next to its histidine kinase.
• the search for histidine kinase is therefore a key step to understanding the signal transduction pathway which leads to the activation of 2C2 (ftcR).
• 23 histidine kinases have been predicted in the genome. It is therefore interesting to mutate them or to use them in a double hybrid screen. Once this kinase has been discovered, a general diagram of the expression of these two proteins and of phosphorylation can be established as a function of growth in liquid culture.
• VjbR is also necessary for the induction of the fliF promoter ( Figure 7) and for the expression of FlgE and FliC. TetR also seems to have an effect on the activation of the fliF promoter ( Figure 5), but this requires confirmation.
• the Brucella sigma factors that encoded by the ORF BMEI0371 is also involved in the regulation of the expression of FlgE and FliC. The deletion of this ORF indeed leads to an overexpression of these two proteins. It is hypothesized that this sigma factor regulates a flagellar repressor. The stage of the flagellar cascade at which this sigma factor intervenes remains to be determined.
• This ORF is surrounded by a histidine kinase (BMEII0370) and a response regulator of two-component systems (BMEII0372) whose role in the expression of flagellar genes must be tested.
• BMEII0370 histidine kinase
• BMEII0372 response regulator of two-component systems
• the initial use of the murine protection model will make it possible to analyze more conditions before possibly selecting certain mutants which will be analyzed subsequently in an infection model using the host species: sheep.
• the coding sequence chosen is the flgl gene encoding a homolog of the flag ring P ring monomer, a ring located in the outer membrane and forming part of the basal body of a flagellar structure, number coding sequence in the genome of B. melitensis 16M: BMEII1084 (Coding sequence of 1074 nucleotides which encodes a predicted protein of 358 residues).
• the mutated strain carries two incomplete copies of flgl.
• PSKKan was obtained by cloning the gene conferring resistance to kanamycin (amplified by PCR from the plasmid pUC4K from Pharmacia with the primers kanamont (5 '-GGGCATGCGGAAAGCCACGTTGTGTCT-3') and kanaval (5 '-GGGGTTACACTAGAA) ) at the Seal site of the pSK-oriT -bla gene.
• PSK-oriT is a derivative of pBluescriptSK (-) (Stratagene) described in Tibor et al, 2002, Infect and Immun, 70: 5540-6. The origin of replication fl of pBluescript was excised by Sspl restriction and replaced by a 0.76 kb Smal-SalI fragment containing the origin of RK2 transfer.
• the vector pSKKAN containing the internal fragment insert of flgl was transferred from Escherichia coli S17-1 to B. melitensis 16M NalR by conjugation.
• the vector is integrated into the Brucella genome, in the flgl gene, leading to two incomplete copies of this gene. Integrating candidates are resistant to kanamycin.
• the site-specific integration was verified by Southern blot on the genomic DNA of integrating candidate clones restricted by HindIII with a probe corresponding to the gene conferring resistance to kanamycin. The known modification of the properties of the host, B.
• melitensis 16M is an attenuation in the murine BALB / c model. Its foreseeable modification is its inability to express the flagellar proteins which are part of the outermost structures (in particular flagellin) and its inability to build a flagellar structure: we expect an unflagellated bacterium. Tests on cellular models in vitro
• the plates are washed and incubated with an additional culture medium comprising 50 ⁇ g of gentamicin (Life Technologies) per ml to kill all the extra-cellular Brucella cells.
• the number of viable intracellular Brucella strains is determined after two hours or 48 hours of post-infection. The data are the average of three culture media and represent three experiments. Flgl flagellar mutants of B. meli tensis did not show any significant attenuation in these models of cell line infection.
• mice Eight week old female mice are inoculated intraperitoneally with 0.1 ml of a suspension comprising 5 ⁇ 10 4 CFU of each bacterial strain (mutant flgl and 16M NalR of parent strain) cultured in the presence of PBS (each dose being confirmed retrospectively). After 8 and 12 weeks of inoculation, 5 mice from each treatment group are sacrificed. The survival rate of the bacteria is determined after homogenization of the mouse spleens in 2 ml of distilled water. Serial dilutions of the homogenates are cultured on TSA to determine the bacterial concentrations.
• HeLa cells were infected with Brucella carrying the plasmid with the GFP reporter under the control of the fliF promoter and observed & after 24 h (A) or 48 hours (B) of infection. Labeling of S-LPS with an antibody detected by red fluorescence was also carried out on cells infected for 24 hours and demonstrates that all bacteria express GFP
• a strain of B. meli tensis 16M carrying a plasmid or the GFP gene is under the control of the fliF promoter was used, while these bacteria cultivated on a bacteriological medium are non-fluorescent. They express GFP after 24 and 48 hours of infection of HeLa cells (FIG. 1a, 1b). At 24 hours, the expression of GFP and therefore the activity of the fliF promoter concerns almost all bacteria, as shown by the immunodetection of S-LPS from Brucella (FIG. These first data prove that the flagellar system is not cryptic and that it is required for the infectious cycle. On the other hand, at this stage, the hypothesis of reconversion into a type III secretion system remains posed.
• Groups of BALB / c mice were infected intraperitoneally (IP) or with WT B. meli tensis 16M strain either by the flagellar mutants corresponding to the MS ring (fliF), to the P ring (flgl) and to the filament (fliC), which corresponds to the three levels of the flagellar structure.
• flagellar CDSs are not exclusively converted into a secretion system since CDSs encoding flagellin (FlgC), the elbow monomer (FlgE) and the P ring (Flgl) are involved in the infectious cycle .
• Another flagellar CDS (FlgF, stem monomer) is also involved in the infectious cycle, it was isolated during an STM screen performed in goats. Tests on pregnant sheep
• PCR polymerase chain reaction
• oligonucleotide primers upflgl 5'-GCGCGCCTGAAGGACATC-3 ') (SEQID N ° 52) and lowflgl (5' -ACGGCGGTCGTGAAATCG- 3 ') (SEQID No. 53) of a fragment going from nucleotide 113 to nucleotide 635 of the coding phase flgl and here called amplicon flgl.
• the flgl amplicon with a size of 533 base pairs was cloned at the EcoRV site of the vector pSKKan, generating the vector pSKKan-flgl.
• the pSKKan was obtained by cloning the gene conferring resistance to kanamycin (amplified by PCR from the plasmid pUC4K from Pharmacia with the primers kanamont (5 '-GGGCATGCGGAAAGCCACGTTGTGTCT-3') (SEQID
• PSK-oriT is a derivative of pBluescriptSK (-) (Stratagene) described in Tibor et al, 2002 (Infect and Immun, 70: 5540-6).
• the origin of replication fl of pBluescript was excised by Sspl restriction and replaced by a 0.76 kb Smal-SalI fragment containing the origin of RK2 transfer.
• the sequence of the amplicon flgl is the sequence SEQID No. 56.
• the amplicon flgl encoding a homolog of the flag ring P ring monomer, ring located in the outer membrane and forming part of the basal body of 'a flagellar structure (coding sequence number in the genome of B. melitensis 16M: BMEII1084).
• This coding sequence of 1074 nucleotides encodes a predicted protein of 358 residues.
• the mutated strain carries two incomplete copies of flgl (SEQID No. 57).
• sequence of the two incomplete copies is the sequences SEQID No. 58 and SEQID No. 59.

Landscapes

• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Health & Medical Sciences (AREA)
• General Health & Medical Sciences (AREA)
• Biochemistry (AREA)
• Biophysics (AREA)
• Life Sciences & Earth Sciences (AREA)
• Genetics & Genomics (AREA)
• Medicinal Chemistry (AREA)
• Molecular Biology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Gastroenterology & Hepatology (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Peptides Or Proteins (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)

Priority Applications (1)

Applications Claiming Priority (3)

Publications (1)

Family

ID=33427305

Family Applications (2)

Family Applications Before (1)

Country Status (2)

Family Cites Families (3)

• 2003
  • 2003-07-03 EP EP03447178A patent/EP1493813A1/de not_active Withdrawn
• 2004
  • 2004-07-05 WO PCT/BE2004/000099 patent/WO2005003353A1/fr active Application Filing
  • 2004-07-05 EP EP04737682A patent/EP1664302A1/de not_active Withdrawn

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events