EP2828376A1 - Procede d'attenuation d'une bacterie du complexe mycobacterium tuberculosis pour la fabrication d'un vaccin contre la tuberculose - Google Patents
Procede d'attenuation d'une bacterie du complexe mycobacterium tuberculosis pour la fabrication d'un vaccin contre la tuberculoseInfo
- Publication number
- EP2828376A1 EP2828376A1 EP13729975.6A EP13729975A EP2828376A1 EP 2828376 A1 EP2828376 A1 EP 2828376A1 EP 13729975 A EP13729975 A EP 13729975A EP 2828376 A1 EP2828376 A1 EP 2828376A1
- Authority
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- European Patent Office
- Prior art keywords
- strain
- tuberculosis
- mycobacterium
- mspa
- vaccine
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
- A61P31/06—Antibacterial agents for tuberculosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
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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
- C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/36—Adaptation or attenuation of cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
- A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/32—Mycobacterium
Definitions
- Tuberculosis is an infectious disease of humans and animals caused by one of the species of the Mycobacterium tuberculosis complex, namely Mycobacterium tuberculosis, Mycobacterium bovis (and the BCG strains derived from it), Mycobacterium africanum, Mycobacterium canettii, Mycobacterium caprae, Mycobacterium microti, and Mycobacterium pinnipedii [19] and Mycobacterium mungii (20). Tuberculosis causes about 9 million new cases worldwide each year and kills about 3 million people each year.
- Tuberculosis is a contagious disease from a person with pulmonary tuberculosis bacillifera, that is to say a person expectorant mycobacteria Mycobacterium tuberculosis complex living in its sputum.
- BCG Bacille Calmette and Guérin
- such attenuated strains of Mycobacterium responsible for infection in humans or animals are suitable for use in the manufacture of vaccines, especially by administering said attenuated bacteria in said vaccine.
- the mycobacterial strains used as vaccines have a survival time in eukaryotic cell culture and in particular in vivo, of not more than 30 days.
- the decrease in growth is accompanied by a decrease in the life of said bacterium in said cells and in particular in vivo MspA porine was found in the membrane extracts of M. smegmatis as an oligomeric protein composed of 20 kDa subunits [5]. It has been considered the major and most abundant porine in M. smegmatis [5, 6]. Suppression of the mspA gene coding for MspA reduces the permeability of the outer membrane of M.
- MspA porine increases the growth of a M.
- the object of the present invention was to provide new M. tuberculosis complex strains with decreased growth rates while maintaining their immunogenicity.
- the inventors have surprisingly found that it is possible to attenuate a bacterium.
- Mycobacterium tuberculosis complex by adding and expressing a gene using molecular engineering methods.
- the present invention provides a method for decreasing the growth of a mycobacterium strain of Mycobacterium tuberculosis complex in eukaryotic cells by adding and expressing in said mycobacterium a mspA gene coding for a porin A of Mycobacterium smegmatis. for use as a vaccine.
- a mycobacterium strain of Mycobacterium tuberculosis complex in eukaryotic cells by adding and expressing in said mycobacterium a mspA gene coding for a porin A of Mycobacterium smegmatis. for use as a vaccine.
- a mspA gene coding for a porin A of Mycobacterium smegmatis. for use as a vaccine.
- tuberculosis and therefore unsuitable for use as a vaccine, and reported reduced viability of a so-called transformed MspA-expressing strain in murine macrophages because said murine macrophages were infected with a leukemia virus murine (MuLV), namely RAW 264.7 cells [28].
- MuLV leukemia virus murine
- Mycobacterium tuberculosis infecting said cells [29]. Therefore, the authors do not mention the interest of a possible use of such a mutant Mycobacterium tuberculosis to confer increased immunity in its host to constitute a vaccine against tuberculosis.
- the present invention provides the use of a Mycobacterium tuberculosis mycobacterium strain, wherein an mspA gene capable of expressing a porin A of Mycobacterium smegmatis has been inserted, for the manufacture of a vaccine for preventing infection by a bacterium of the Mycobacterium tuberculosis complex in a host comprising eukaryotic cells, preferably macrophages, said strain of mycobacteria thus transformed having decreased growth in said eukaryotic cells.
- the present invention relates to the use of a Mycobacterium tuberculosis transformed strain according to the invention for the manufacture of a vaccine for the prevention of tuberculosis in mammals or birds, said cells being macrophages.
- the subject of the present invention is a use of a transformed strain according to the invention which is useful for the manufacture of a vaccine for the prevention of human tuberculosis.
- a strain thus transformed of mycobacterium of the Mycobacterium tuberculosis complex allowing the expression of MspA porine in a said mycobacterium of the M. tuberculosis complex has a greater growth than the wild-type strain.
- Mycobacterium tuberculosis in culture medium without cells eukaryotes (axenic medium) - in this the inventors have confirmed the results previously published in the literature [12, 14]; b- but, surprisingly, the growth of this transformed strain, in co-culture of eukaryotic cells, makes it possible to reduce the growth of the mutant Mycobacterium tuberculosis compared to that of the original wild-type strain and this by using three mediums of different eukaryotic cell cultures, including in particular mouse macrophages and human macrophages.
- these macrophages are particularly concerned in tuberculosis and immunogenicity reactions and therefore protection in use as a vaccine.
- macrophages are the cells of humans and animals that phagocytize Mycobacterium tuberculosis mycobacteria in an attempt to destroy them and to present mycobacterial antigens to other cells of the immune system [25].
- said mspA gene placed under the control of a promoter allowing the expression of said mspA gene in said mycobacterium of the Mycobacterium tuberculosis complex is inserted into said strain. More particularly, a plasmid containing said mspA gene is introduced under the control of a promoter of the expression of said mspA gene in a said bacterium of the Mycobacterium tuberculosis complex.
- said mspA gene and said control elements for the expression of said mspA gene in a said bacterium of the Mycobacterium tuberculosis complex are inserted into the chromosome of said bacterium of the Mycobacterium tuberculosis complex.
- bacteria of the Mycobacterium tuberculosis complex is intended here to mean one of the bacteria of the species chosen from at least Mycobacterium tuberculosis, Mycobacterium bovis and its clones or subspecies BCG, Mycobacterium africanum, Mycobacterium canettii, Mycobacterium caprae, Mycobacterium microti, Mycobacterium pinnipedii, and Mycobacterium mongii. These species are all neighbors with more than 99% similarity of their genome for the species currently sequenced; they all induce the same clinical disease and cause the same tissue lesions in the infected organs, characterized by the formation of granulomas with macrophagic giant cells [26].
- said strain of the Mycobacterium tuberculosis complex is a strain of the Mycobacterium tuberculosis species.
- said strain of Mycobacterium tuberculosis of origin is the H37Rv strain.
- said mspA gene comprises the sequence SEQ ID No. 1 of the sequence listing appended to the present description.
- said mspA gene is under the control of expression elements called "hsp60 promoter" consisting of SEQ. ID. No. 4 of the sequence listing appended to this description.
- said eukaryotic cells are mammalian or bird cells, preferably said cells are human cells. More particularly, said cells are macrophage cells.
- the present invention also provides a vaccine comprising a transformed strain of a bacterium of the Mycobacterium tuberculosis complex into which a said mspA gene capable of expressing porin A of Mycobacterium smegmatis has been inserted into a so-called mycobacterium making it possible to implement a method according to the invention.
- H37RvpVVMspA the strain, hereinafter referred to as H37RvpVVMspA, filed on April 26, 2012 at the National Collection of Type Cultures (NCTC) collection, Health Protection Agency Culture Collections, Porton Down, Salisbury Wiltshire, SP4 OJG UK under number 12042601. This deposit was made in accordance with the Budapest Treaty.
- a transformed strain according to the invention more particularly exhibits a decrease in the growth of said bacterium in eukaryotic cells by at least 90%, or even at least 99% in terms of the number of bacteria per ml of cell culture.
- the number of bacteria per ml of cell culture is divided by at least 10, or even divided by at least 100.
- a strain of mycobacterium transformed according to the invention has a survival time in culture of eukaryotic cells and in particular in vivo, not more than 60 days, preferably not more than 50 days.
- the decrease in growth is accompanied by a decrease in the life of said bacterium in said cells and in particular in vivo of at least 3 to 10 days.
- FIG. 1 represents an SDS-PAGE gel photograph showing the level of expression of MspA porine.
- Lane M shows the molecular weight markers.
- Lane 1 represents the separation of a protein extract from M. smegmatis
- Lane 2 a protein extract from M. tuberculosis H37Rv / pVV16
- Lane 3 represents the separation of a protein extract from M. tuberculosis H37Rv / pVVMspA, the MspA protein being apparent at 20 KDa.
- FIG. 2 represents the growth curves of M. tuberculosis growth in the presence of MspA porine on 7H9 axenic media of Example 1, measured in optical density (OD) ordinates at 600 nm, from 0 to 1, for 18 days ("t" on the abscissa).
- FIGS. 3A to 3C show the intracellular growth graphs of M. tuberculosis H37Rv / pVV16 and M. tuberculosis H37Rv / MspA in various eukaryotic cells: A. polyphaga (FIG. 3A), BMDMs (FIG. 3B) and hMdMs (FIG. 3C) Examples 2 and 3, measured in number of CFU / ml of cell lysate, from 0 to 1,000,000 (on the ordinate) after 0 to 14 days of cultures ("t" on the abscissa).
- FIG. 4 represents the evolution of the weight of the mice (w) in grams (g) as a function of time (t), in number of days, from 0 to 39, for mice infected with the wild-type strain H37Rv and for mice infected with modified strain H37Rv / pVVMspA at different concentrations (10,000 CFU / 100 ⁇ and 10,000 CFU / 100 ⁇ ).
- FIG. 5 represents the bacterial load (number of CFUs) in the liver (A), the spleen (B) and the lung (C), after a inoculation with the wild-type strain of M. tuberculosis H37Rv ( ⁇ ) and the strain of M. tuberculosis according to the present invention ( ⁇ ), as discussed in Example 4.
- Example 1 Growth of Mycobacterium tuberculosis expressing MspA in axenic medium.
- the inventors amplified the gene encoding MspA porine from the genome of Mycobacterium smegmatis, and then introduced the amplification product into the expression vector pVV16 [16] which is characterized by the presence of two genes coding for hygromycin and kanamycin resistance proteins (selection markers) to obtain plasmid pVVMspA.
- the inventors incorporated plasmid pVVMspA by electroporation into Mycobacterium tuberculosis strain H37Rv and the selection of transformed clones was performed using hygromycin and kanamycin as antibiotics.
- the inventors extracted the genomic DNA of the strain M. smegmatis mc 2 155 (ATCC 700084) and amplified by standard PCR the gene encoding porin MSpa [5].
- sequence gi_118467340 of said gene is the sequence SEQ. ID. n ° following:
- the primers used are:
- MSPA-pVV16F 5'-ccccccatatg_aaggcaatcagtc-3 '(SEQ ID NO: 2), and
- MSPA-pVV16ndeI 5'-cccatatg_tcagttcatgttccaggg-3 '(SEQ ID NO: 3).
- primers were determined to generate a PCR product corresponding to the entire mspA gene (636 bp) harboring on each side a cleavage site for the restriction enzyme Nde I (this enzymatic cleavage site is underlined in the sequences of primers above) to allow cloning into the pVV16 expression vector; then its expression in a constitutive way.
- the pVV16 vector has a hygromycin resistance cassette and an expression hsp60 promoter (SEQ ID NO: 4) in mycobacteria [16].
- the specificity of pVV16 is an expression promoter in Escherichia coli (used in the verification of plasmid construction at the time of cloning) and a kanamycin resistance cassette allowing for double selection [16].
- the PCR fragment was cloned into the NdeI site of the plasmid pVV16 to give the plasmid pVVMspA.
- the latter was introduced into Escherichia coli DH5 alpha by the simplest means: thermal shock.
- the plasmid construction was verified by enzymatic digestion and amplification by PCR and sequencing.
- the sequence SEQ. ID. # 4 of the hsp60 promoter is 5'-
- the expression cassette of the mspA gene under the control of the hsp60 promoter consists of the juxtaposition of SEQ. ID. No. 4 and SEQ. ID. No. l.
- Mycobacterium tuberculosis strain H37Rv (ATCC 27294) [17] electrocompetent mycobacteria (permeable to introduction of a plasmid) were prepared as previously described by Van Kessel and Hatfull [18]. Briefly, Mycobacterium tuberculosis mycobacteria were harvested from a liquid culture of M. tuberculosis H37Rv of optical density (OD 60 o) between 0.8-1.0, washed three times in 10% glycerol and resuspended. in the same buffer.
- OD 60 o optical density
- the plasmids pVV16 control to ensure that the growth changes are not due to the expression vector itself but to the presence of mspA gene
- pVVMspA plasmid containing the MspA d porine gene. interest
- electroporator reference Multiporator / Electroporator 2510, Le Pecq, France
- Electroporated mycobacteria were recovered in 1 ml of Middelbrook 7H9 enriched liquid medium (Sigma-Aldrich, Lyon, Way), cultured at 37 ° C.
- MspA porine is characterized by its stability to heat denaturation [5].
- an SDS-PAGE electrophoresis gel was made from the protein extracts of M. tuberculosis H37Rv / pVV16 (lane 2), M. tuberculosis H37Rv / pVVMspA (lane 3) and M. smegmatis (control positive) (track 1). These extracts were prepared by selective extraction by high temperature [27].
- Figure 1 shows the expression of the mspA gene in M. tuberculosis H37Rv and in the M. smegmatis control strain. Detected protein spots were excised from the gel and analyzed by MALDI-TOF mass spectrometry. Protein stains were identified with a sequence coverage ranging from 21% to 29% and a similarity to the MspA protein sequence of M. smegmatis at 20 KDa.
- M. tuberculosis H37Rv / pVV16 Transformed Control Strains Not Expressing MspA Porine
- M. tuberculosis H37Rv / pVVMspA Transformed Strains Expressing Porina MspA
- M. tuberculosis H37Rv / pVVMspA Transformed Strains Expressing Porina MspA
- 5mL of Middlebrook 7H9 medium Becton Dickinson, Claix's Bridge, Way
- the mycobacteria were strongly mixed with stirring vortexed to remove cell clumps, harvested and diluted in 10 ml of fresh 7H9 medium.
- Growth rates were determined for both strains in three independent cultures by OD 60 measurements in a BACTEC MGIT 960 system (Becton Dickinson, Claix Bridge, Way). This Fully automated system is based on the detection of oxygen consumption from aerobic bacteria.
- MspA protein carried out in vitro growth experiments with M. tuberculosis / pVV 16 and M. tuberculosis / pVVMsp A strains in a 7H9 axenic liquid medium in three independent cultures.
- the inventors used a cell suspension and the growth of the bacteria was measured in the BACTEC MGIT 960 system for 17 days.
- the generation time of the M. tuberculosis / pVV 16 control strain and the M. tuberculosis / pVVMsp A strain were respectively 19h 10min ⁇ 10min and 17h 16min ⁇ 12min ( Figure 2).
- the inventors have thus found that the strain which expresses MspA porine grows significantly faster than the control strain (p ⁇ 0.05). This experiment, performed three times, showed that MspA porine expression increases the growth rate of M. tuberculosis.
- Figure 3A shows the intracellular growth graphs of M. tuberculosis H37Rv / pVV16 and M. tuberculosis H37Rv / MspA in eukaryotic cell culture: A. polyphaga.
- Example 3 Growth of Mycobacterium tuberculosis expressing MspA in co-culture with human macrophages (hMdMs) or mouse macrophages (BMDMs):
- BMDMs [24] and hMdMs isolated from leukopacks (French Blood Establishment, Marseille, Way) by a differentiated Ficoll gradient (MSL, Eurobio, Courtaboeuf, Manière) were seeded separately (10 5 cells / well) in 24-well culture plates in RPMI 1640 medium containing 10% FCS.
- BMDMs and hMdMs were separately infected with M. tuberculosis, M. tuberculosis H37Rv / pVV16, and M.
- tuberculosis H37Rv / pVVMspA for 24 hours, then the macrophages were washed to remove free mycobacteria before being incubated for different periods of time in RPMI 1640 medium containing 10% calf serum (FCS).
- FCS calf serum
- each cell type as well as mycobacteria were cultured separately. Determination of the number of intracellular mycobacteria
- the infected cells were lysed with 0.1% sodium dodecyl sulfate (SDS) for 30 min and then passed through a 26 gauge needle to ensure complete lysis.
- SDS sodium dodecyl sulfate
- the lysate (500 ⁇ ) was washed three times with PBS, plated on solid 7H10 medium (Becton Dickinson, Claix's Bridge, Way) and incubated for 15 days at 37 ° C to determine the number of colonies of intracellular mycobacteria ( UFC). All experiments were conducted in triplicate.
- BMDM macrophages as eukaryotic cells, the inventors observed that both M. tuberculosis strains H37Rv / pVV16 and M. tuberculosis H37Rv / pVVMspA survived throughout the time of the experiment (14 days). The inventors observed that between the 4 th and the 14 th day, the H37Rv / pVV16 control strain was significantly higher than the strain that expresses MspA (p ⁇ 0.05) (FIG. 3B). To I4 th day, the number of colonies was 1 ⁇ 10 6 ⁇ 7 ⁇ 10 5 M. tuberculosis H37Rv / pVV16 against 4 ⁇ 10 5 ⁇ 1 May 10 in M. tuberculosis H37Rv / pVVMspA (p ⁇ 0.05) .
- Figures 3B and 3C show the intracellular growth graphs of M. tuberculosis H37Rv / pVV16 and M. tuberculosis H37Rv / MspA in different macrophage cultures: BMDMs (fig.3B) and hMdMs (fig.3C).
- Example 4 Growth of Mycobacterium tuberculosis expressing MspA in animal model and protection against tuberculosis
- mice 6 to 8 weeks old (10 per group) were infected intraperitoneally with 100 ⁇ l of bacterial culture.
- Two different inoculums of each of the above strains wild strain H37Rv ATCC 27294 and mutated strain M. tuberculosis H37Rv / pVVMspA were used: 1 X 10 7 CFU / mL and 1 X 10 5 CFU / mL.
- As a control one group of mice was infected with 100 ⁇ l of sterile PBS. The weight, performance and survival of the mice were monitored for 1 month.
- mice The examination of the infected mice was carried out according to a protocol published by other researchers [3]. Also, at first some surviving mice were sacrificed, the spleen, liver and lungs were analyzed for pathological anatomy and the load Bacterial was determined per gram of tissue, per culture and expressed in CFU. In a second step, the surviving mice of both groups were re-inoculated with 10 7 CFU / mL M. tuberculosis h37Rv and the same observation protocol was followed.
- mice infected with the strain modified according to the invention have less behavioral problems and grow faster than mice infected with the wild strain of M. tuberculosis ( Figures 4 and 5).
- mice inoculated with the mutated strain M. tuberculosis H37Rv / pVVMspA survived, while those inoculated with the wild-type strain showed 50% mortality. This survival was correlated with a very low number of colony forming units (CFU) of M. tuberculosis H37Rv / pVVMspA in the lungs. Mice inoculated with the mutated strain M. tuberculosis H37Rv / pVVMspA developed few tissue lesions with small granulomas. A mouse inoculated with the wild-type M.
- CFU colony forming units
- tuberculosis H37Rv strain presented an abdominal fistula. Macroscopic analysis showed that the spleen weights of mice inoculated with the wild-type M. tuberculosis H37Rv strain was significantly greater than that of mice inoculated with the mutated strain. Also, the number of granulomas was respectively in the liver 24 ⁇ 21 vs 0; and in the spleen 61 ⁇ 27 vs 8 ⁇ 7, these differences being significant (p ⁇ 0.05). In the spleen, enumeration of mycobacteria showed a significant decrease in the number of CFU for the mutated strain M. tuberculosis H37Rv / pVVMspA compared to the wild-type strain M. tuberculosis H37Rv. The immunogenicity tested in the cell assays was retained.
- mice 90 days after the first inoculation, the inventors inoculated again the two groups of mice with the wild strain of Mycobacterium tuberculosis H37RV using an inoculum 10 7 CFU per ml. The mice were again followed for 90 days, all mice survived with the exception of one in four mice inoculated with the wild strain during the first inoculation.
- mice were euthanized 90 days after the second inoculation, the histopathological analysis of the tissues showed a significantly larger number of pulmonary granuloma nodules in the group initially inoculated with wild-type tuberculosis (33 ⁇ 37 nodules than in the initially inoculated mice). with the vaccine strain according to the present invention (21 ⁇ 25 nodules) (p ⁇ 0.05).
- the inventors thus obtained three types of evidence, a clinical evidence (mortality); histological evidence (number of granulomas per organ); and a microbiological evidence (bacterial load after inoculation) to conclude that mutated strain M. tuberculosis H37RV / PVVMSPA confers protection in this animal model against wild-type tuberculosis.
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Abstract
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Application Number | Priority Date | Filing Date | Title |
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FR1254623A FR2990697B1 (fr) | 2012-05-21 | 2012-05-21 | Procede d'attenuation d'une bacterie du complexe mycobacterium tuberculosis pour la fabrication d'un vaccin contre la tuberculose |
PCT/FR2013/051078 WO2013175103A1 (fr) | 2012-05-21 | 2013-05-16 | Procede d'attenuation d'une bacterie du complexe mycobacterium tuberculosis pour la fabrication d'un vaccin contre la tuberculose |
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EP13729975.6A Withdrawn EP2828376A1 (fr) | 2012-05-21 | 2013-05-16 | Procede d'attenuation d'une bacterie du complexe mycobacterium tuberculosis pour la fabrication d'un vaccin contre la tuberculose |
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US (1) | US20150157701A1 (fr) |
EP (1) | EP2828376A1 (fr) |
JP (1) | JP2015518719A (fr) |
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US20220361461A1 (en) * | 2019-06-28 | 2022-11-17 | United States Government As Represented By The Department Of Veterans Affairs | Methods of producing granulomas and animal models of sarcoidosis |
CN113493788B (zh) * | 2021-05-24 | 2023-06-23 | 上海晶诺生物科技有限公司 | 一种分枝杆菌启动子库及其应用 |
CN118240732A (zh) * | 2024-04-01 | 2024-06-25 | 华中农业大学 | 一种结核分枝杆菌Rv3444-Rv3445双基因缺失菌株及其制备和应用 |
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FR2697022B1 (fr) * | 1992-10-19 | 1994-12-16 | Pasteur Institut | Antigènes de Plasmodium falciparum capables d'induire des anticorps protecteurs à large spectre - Application à la vaccination. |
US20050208078A1 (en) * | 2003-11-20 | 2005-09-22 | Hoffman Stephen L | Methods for the prevention of malaria |
EP3686602A1 (fr) * | 2008-09-22 | 2020-07-29 | University of Washington | Nanopores msp et procédés associés |
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Publication number | Publication date |
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CN104271732A (zh) | 2015-01-07 |
FR2990697B1 (fr) | 2015-12-18 |
JP2015518719A (ja) | 2015-07-06 |
US20150157701A1 (en) | 2015-06-11 |
FR2990697A1 (fr) | 2013-11-22 |
WO2013175103A1 (fr) | 2013-11-28 |
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