WO2012016315A1 - Souche recombinante de la bactérie brucella spp et vaccin vivant contre la brucellose - Google Patents

Souche recombinante de la bactérie brucella spp et vaccin vivant contre la brucellose Download PDF

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WO2012016315A1
WO2012016315A1 PCT/BR2011/000276 BR2011000276W WO2012016315A1 WO 2012016315 A1 WO2012016315 A1 WO 2012016315A1 BR 2011000276 W BR2011000276 W BR 2011000276W WO 2012016315 A1 WO2012016315 A1 WO 2012016315A1
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pgk
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delta
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WO2012016315A4 (fr
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Sérgio COSTA OLIVEIRA
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Universidade Federal De Minas Gerais - Ufmg
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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
    • 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)
    • C12N9/1217—Phosphotransferases with a carboxyl group as acceptor (2.7.2)
    • 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
    • A61K39/098—Brucella
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/02—Phosphotransferases with a carboxy group as acceptor (2.7.2)
    • C12Y207/02003—Phosphoglycerate kinase (2.7.2.3)
    • 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

Definitions

  • the present invention belongs to the fields of immunology and molecular biology / microbiology, in particular bacteriology. More specifically, this invention describes the development of a recombinant Brucella spp. Strain and its use as a vaccine for the control of Brucellosis disease in mammals.
  • Brucellosis is a zoonosis caused by bacteria of the genus Brucella, which infect humans and domestic animals (NICOLETTI, PL Relationship between animal and human disease. In: Brucellosis: clinical and laboratory aspects. Young, EJ and Corbel, MJ. CRC Press, Inc., Boca Raton, FL, p.41-51, 1989). Brucellosis is considered by WHO as the most widespread of all zoonoses. This disease brings two concerns: sanitary, due to its possibility of transmission to man; and economic, due to decreased animal productivity, invalidation of products and derivatives as a result of this disease, trade restrictions, animal discarding, increased replacement rate of animals, calf death and increased calving interval (FRENEY, J RENAUD, F.
  • brucellosis causes losses of 25% in milk and meat production and 15% in calf production (BRAZIL. Ministry of Agriculture, Livestock and Supply. Epidemiological situation of bovine and buffalo brucellosis in Brazil - First partial report 2006. 83p).
  • the control of human brucellosis is based on vaccination, diagnosis and elimination of infected animals, as well as food hygiene measures. All attempts at human vaccination have still been shown to be ineffective and dangerous, and there is a need to develop a truly effective human vaccine to control this disease (Freney et al, 2000).
  • the S19 vaccine strain developed in the 1920s for bovine immunization, is attenuated and has a natural deletion in the EryC gene that is required for erythritol metabolism. This strain induces about 60% protection against infection and protects about 70% of animals for 4-5 pregnancies against abortion.
  • Both B. abortus strain S19 and B. melitensis strain Rev 1 are effective in protecting against their respective pathogens, but these vaccine strains have three major disadvantages: they are pathogenic to humans and can cause miscarriage when administered to pregnant females.
  • the RB51 strain has a modification in the structure of the LPS, since it does not have the O-antigen, since it has a mutation in the wboA gene, which encodes a glycosyl transferase involved in N-formylperosamine polymerization (MORIY ⁇ N, I .; GRILL ⁇ , MJ; MONREAL, D.; GONZALEZ, D .; MARIN, C.; L ⁇ PEZ-GONI, I.; MAINAR-JAIME, RC; MORENO, E.
  • This strain is more sensitive to complement-mediated lysis (UGALDE, JE; CZIBENER, C; FELDMAN, MF & UGALDE, RA. Identification and characterization of the Brucella abortus phosphoglucomutase gene: 68, p 5716-5723, 2000).
  • the rough strain has the advantage that it is less virulent and less abortive than the smooth vaccine strain and does not interfere with the differential diagnosis between vaccinated and infected animals.
  • the main disadvantage of RB51 is that it is resistant to the antibiotic rifampicin, which is widely used in combination with other antibiotics in human treatment (WHO, 1998).
  • WO9937783 Live vaccine against brucellosis - describes recombinant Brucella strains constructed by deletion of the Brucella rfbU gene. This recombinant strain is attenuated and induces protective immunity similar to that induced by commercial strains.
  • CN 101092605 Mutant strain of Brucella bacterin with weak poison, constructing method, and application - describes recombinant Brucella strains constructed by deletion of the Brucella S19, Bp26, wboA, omp31, and P39 genes. These strains induce immunity. have the same virulence as the commercial vaccine and are unable to interfere with the diagnosis of infected individuals or populations.
  • CN101386833 Recombinant bacterium of Brucella abortus and use thereof - describes recombinant Brucella strain constructed by deletion of the gene coding for Brucella S19 cold shock protein. This recombinant strain is attenuated, has less virulence than the commercial strain and is unable to interfere with the diagnosis of infected individuals or populations.
  • CN101386831 Recombinant bacterium of Brucella abortus with immunity labeling and use thereof - describes recombinant Brucella strain constructed by deletion of the gene coding for Brucella S19 perosamine synthase enzyme. This recombinant strain is attenuated, has less virulence than the commercial strain and is unable to interfere with the diagnosis of infected individuals or populations.
  • the present invention involves the development of a recombinant strain of Brucella S2308 bacterium that has partial or complete deletion of the pgk gene encoding the phosphoglycerate kinase (PGK) enzyme.
  • PGK is an important glycolytic pathway enzyme that reversibly catalyzes the transfer of a 1,3 bisphosphosphoglycerate phosphate to ADP, thereby forming 3-phosphoglycerate and ATP (BERNSTEIN, BE; MICHELS, PA & HOL, WG Synergistic effects of substrate-induced conformational changes in phosphoglycerate kinase activation Nature, v. 385 (6613), pp. 275-8, 1997).
  • bacteria with deletion of this gene are attenuated and induce higher protective immunity than that induced by commercial strains.
  • B. abortus strain S19 and B. melitensis strain Rev 1 are effective against their respective pathogens, but these vaccine strains have three major disadvantages: they are pathogenic to humans, can cause miscarriage when administered to pregnant females and induce antibody production in immunized animals, which interferes with the diagnosis of infected individuals or populations (Cheville et al., 1992, 1993 and 1996).
  • the RB51 rugosa strain has the advantage that it is less virulent than the smooth vaccine strain, less abortive than the other vaccine strain and not interfere in the differential diagnosis between vaccinated and infected animals.
  • the main disadvantage of RB51 is that it is resistant to the antibiotic rifampicin, which is widely used in combination with other antibiotics in human treatment (WHO, 1998).
  • Recombinant strain S2308Apg / from Brucella abortus S2308 is capable of inducing protective immunity in 129 / Sv and IRF-1 KO mice higher than commercial strains S19 or RB51, and protection equal to strain S19 in BALB / c and C57BL / 6 mice, presenting potential for use as a new live brucellosis vaccine.
  • a major advantage of strain S2308Apgk is that it has a defined and known genetic alteration.
  • the S2308Apg strain is much more attenuated than the S19 smooth strain.
  • Figure 1 Western blot analysis of pgk expression in B. abortus S2308 (channel 1), S2308Apg / (channel 2) and S2308Apgr / c complemented with pBBR1-pgk (channel 3). The presence of approximately 42 kDa band in channels 1 and 3 indicates pgk expression in B. abortus S2308 and S2308Apgr f strains complemented with pBBR1-pgk.
  • Figure 2 Intracellular replication of mutant strains S230SApgk and S " ⁇ 9Apgk in bone marrow-derived macrophages. Adherent cells were infected with B.
  • FIG. 3 Susceptibility of IRF-1 " ' " mice inoculated with mutant strains S2308Apgfr and S19Apgk, vaccine strains RB51 and S19 and virulent strain B. abortus S2308. Eight mice per group were infected with a dose of 1x10 6 CFU. The mice were followed daily for 28 days.
  • FIG. 4 Protection of IRF-1 " ' " mice against challenge with B. abortus virulent strain S2308 after immunization with strain S2308Apgfr, S Sàpgk, RB51 and S19.
  • Mice were immunized intraperitoneally with a dose of 1x10 5 CFU of each strain separately, except strain RB51 which were vaccinated with 1x10 7 CFU. Twelve weeks later they were challenged in the same way with 1x10 6 CFU of the virulent strain S2308. The mice were followed daily for 30 days.
  • FIG. 5 Persistence of the S19Apg / re mutant strain of the S19 vaccine strain in C57BL / 6 (A) and 129 / Sv (B) mice. Eight mice per group were infected with a dose of 1x10 6 CFU of each strain separately. Spleens were collected 1, 2, 3, 4 6 weeks after infection and the number of CFUs was determined in macerated tissues by serial dilution and plating. Values are expressed as mean CFU. The asterisks represent the statistically significant difference from the S19Apgr / group compared to the group that received the B. abortus S2308 strain (p ⁇ 0.05).
  • the recombinant strain of Brucella bacterium characterized by having the pgk gene modified by partial or complete deletion is obtained by introducing a plasmid containing the pgk gene interrupted by a fragment containing a kanamycin antibiotic resistance gene. For this, the following detailed methodology was used.
  • B. abortus S2308 genomic DNA was performed according to Halling et al. (1991), with some modifications. From a stock aliquot of S2308, a striation was made on BB / Agar medium. The plates were incubated for 72 hours in a 37 ° C oven containing 5% CO 2 . An isolated colony was inoculated into 2 ml BB medium and incubated at 37 ° C under 200 rpm shaking for 48 hours. After this time the culture was inactivated for 1 hour at It was then centrifuged at 7000 rpm for 10 minutes and the pellet was resuspended in 1.5 ml of TE.
  • the mixture was incubated on ice and gently shaken for 30 minutes, and then centrifuged at 7000 rpm for 10 minutes.
  • the aqueous phase recovery process was repeated twice. After the last repetition, the same volume of phenol-chloroform (1: 1) was added, the mixture was incubated on ice and gently shaken for 30 minutes, and then centrifuged at 7000 rpm for 10 minutes.
  • the aqueous phase was again recovered and 0.6 mL of isopropanol was added to it so that genomic DNA precipitated. After DNA precipitation, it was removed and washed twice with 70% ethanol, then dried at room temperature and resuspended in 100-200 ⁇ of sterile milli-Q water.
  • the quality and concentration of DNA was evaluated by 0.8% agarose gel electrophoresis and spectrophotometer analysis at 260 and 280 nm wavelengths (UV mini 1240 UV-VIS Spectrophotometer Shimazdu).
  • the pgk gene was amplified from B. abortus genomic DNA.
  • 10ng of genomic DNA, 1 ⁇ of each primer ( ⁇ pmoles ⁇ L), 1 ⁇ _ of Taq DNA 10X concentrated polymerase buffer (500mM Tris-HCI pH 9.0 and 1% Triton X-100) were used.
  • 0.8 ⁇ _ 25mM MgCl 2 0.25 ⁇ _ 10mM dNTPs
  • the PCR reaction was performed following the following program:
  • the product was submitted to 0.8% agarose gel electrophoresis and the fragment of interest was purified from the gel by the Wizard SV Gel and Clean-Up PCR System kit (Promega), according to the manufacturer's guidelines. Soon after, the fragment was dosed on 0.8% agarose gel with DNA fragments of known concentration.
  • Amplified product and plasmid pBluescript-KS (+) were double-digested in separate tubes with restriction enzymes Bam H1 and Kpn I. Following digestion, samples were subjected to 0.8% agarose gel electrophoresis in ethidium bromide stained TAE buffer. The pBluescript-KS (+) vector and 1191 bp fragment of interest were removed from the gel, purified using the Wizard Wizard SV Gel and Clean-Up PCR System kit (Promega), and then electrophoresed with 1 Kb Ladder. (Invitrogen) and DNA of known concentration.
  • the 1191bp fragment was ligated into the pBluescript-KS (+) vector by obeying the 1: 3 vector / fragment molar ratio.
  • 2 ⁇ _ of the 5X binding buffer and 1.5 ⁇ _ of the enzyme T4 DNA Ligase were used. 1 ⁇ / ⁇ _ (Promega) in a final volume of 10 ⁇ _.
  • the ligations were performed for 16 hours at 16 ° C.
  • the ligation product was renamed pBluescript-
  • E. coli DH5a competent cells were prepared by the calcium chloride technique described by Sambrook et al, 1989.
  • 5 ⁇ _ of the vector was used for 100 ⁇ _ of competent cells.
  • the cells were centrifuged, resuspended in 100 ⁇ _ LB and plated on LB agar supplemented with 100 ⁇ g / mL ampicillin, 50 ⁇ g / mL X-gal and 0.5mM IPTG and incubated at 37 ° C for 16 hours. hours
  • White colonies were selected and inoculated in ampicillin-supplemented LB medium (100 ⁇ g / mL). The cultivation was grown for 16 hours at 37 ° C under constant agitation.
  • DNA from recombinant colonies was subjected to restriction analysis with the enzymes Bam HI and Kpn I. Cloning was confirmed by sequencing plasmid DNA from recombinant colonies using the same primers used for pgk gene amplification. The obtained sequences were further analyzed and manually edited to eliminate ambiguities, vector regions and poor quality data at the end of the sequences.
  • the kanamycin resistance conferring gene was obtained from plasmid pUC-4K (GE Healthcare).
  • the enzyme Eco RI was used for ORF extraction of the kanamycin gene from the plasmid.
  • the digestion reaction was performed with the vector according to the enzyme manufacturer's New England Biolabs guidelines. After digestion, the sample was submitted to 0.8% agarose gel electrophoresis. The fragment was removed from the gel and purified by the Wizard SV Gel and Clean-Up PCR System kit (Promega).
  • the enzyme Eco RI was used for cloning the kanamycin gene into the pBluescript-pgr vector.
  • the binding product is renamed pBluescript-pg / c -Kan.
  • Competent E. coli DH5 cells were prepared by the calcium chloride technique described by Sambrook et al. in 1989.
  • 5 ⁇ _ of the vector was used for 100 ⁇ _ of competent cells.
  • the pBluescript: pg // Kan vector was extracted from E. coli DH5a by the alkaline lysis method using the Wizard mini prep kit (Promega). Plasmid quantitation was estimated on 0.8% agarose gel.
  • S2308 and S19 electrocompetent cells were prepared according to Halling et al. (1991), implementing some modifications. From the stock of S2308 and one from S19, striations were made on BB / agar medium, the plates were grown for 72 hours in a 37 ° C oven containing 5% CO 2 . An isolated colony of each strain was inoculated into 2 ml BB medium and incubated at 37 ° C with 200 rpm shaking for 48 hours. After this time, the saturated cultures were added to 200 ml BB medium in 1000 ml conical flask and incubated again at 37 ° C with 200 rpm shaking until reaching ⁇ at about 0.4 to 0.5.
  • the cultures were centrifuged at 7000 rpm for 10 minutes and the pellets were washed with 100 mL sterile milli-Q water. One more centrifugation and washing step was performed. One more centrifugation was performed and the pellets washed with 2 mL of milli-Q water plus 10% glycerol. Another centrifugation was performed and the pellets were resuspended in 500 ⁇ _ of milli-Q water plus 10% glycerol, the cells were immediately used. Electroporation was based on the protocol proposed by Drazek et al, 1995, with some modifications. B. abortus S2308 and S19 electrocompetent cells were placed on ice to thaw slowly.
  • Colonies isolated from the original plate were picked on medium BB / agar plus kanamycin and BB / agar plus ampicillin with sterile tips. The plates were grown for 72 hours at 37 ° C in a 5% CO 2 oven. Clones grown on both selective media were characterized as single recombinant (Kan r Amp r ) and clones grown only on kanamycin containing medium were characterized as double recombinant (Kan r Amp s ). Kan r Amp s strains were selected and colony PCR was performed with primers that amplify the kanamycin gene for initial confirmation of the mutation. Following this initial confirmation, wild-type DNAs were extracted for mutation confirmation by the Southern blot technique.
  • Geno evidence that the wild gene was exchanged for the kanamycin cassette disrupted gene was performed using the Southern blot technique for differentiation between single-recombinant transformants and double-recombinant transformants. Isolation of genomic DNA from recombinant clones was performed according to the methodology described above. Approximately 10 ⁇ g of the genomic DNA of each transformant was subjected to Eco RV restriction enzyme digestion according to the protocol stipulated by the manufacturers. As control we used genomic DNA from B. abortus and S19 parental strains S2308 digested with the same restriction enzyme.
  • Probes were constructed for the pgk gene and the kanamycin and ampicillin resistance genes according to the following table:
  • AlkPhos Direct kit (GE Healthcare) was used to label the fragments obtained for the probe.
  • Membranes were prehybridized (Alkphos Direct Labeling and Detection Systems) for 30 minutes at 65 ° C under moderate agitation in 15 mL of hybridization solution. Labeled probes were added after 30 minutes of prehybridization.
  • Hybridization was performed at 65 ° C for 16 hours in a hybridization oven (Techne hybridiser HB-1D, Techne, Cambridge, U.K.) under gentle agitation.
  • the pgk gene ORF probe hybridized to an approximately 5800 bp fragment for B. abortus S2308 and an approximately 7082 bp fragment to the double recombinant clones, called S2308Apg /.
  • the recombinant single transformant showed a fragment of approximately 10042 bp, which corresponds to the insertion of all the suicide plasmid within its genome. This result was confirmed when the ampicillin gene was used as a probe, where hybridization was only observed in the recombinant single transformant.
  • the probe for the kanamycin resistance gene hybridized to the transformant genomic DNA and did not hybridize to the wild type S2308 genomic DNA.
  • the pgk gene was amplified with the following pair of primers designed from B. abortus sequences:
  • the PCR reaction was performed following the following program:
  • the product was resolved on 0.8% agarose gel and the fragment of interest was purified from the gel by the Wizard SV Gel and Clean-Up PCR System kit (Promega), according to the manufacturer's guidelines. Soon after, the fragment was dosed on 0.8% agarose gel.
  • Amplified product and plasmid pBBR1-MCS were digested with restriction enzymes Kpn I and Bam Hl, purified from agarose gel by Wizard SV Gel and Clean-Up PCR System kit (Promega) and ligated with T4 DNA Ligase enzyme. (Promega). Following ligation, competent E. coli DH5 cells were transformed.
  • DNA from recombinant colonies was subjected to restriction analysis with the enzymes Kpn I and Bam H1 and sequencing using the same primers used in the second amplification of the pgk gene. After confirmation of the correct cloning of the pgk gene in plasmid pBBR1-MCS this gene construct was renamed pBBR1-pgk.
  • the pBBR1-pgk vector was extracted from E. coli DH5a by the alkaline lysis method using the Wizard mini prep kit (Promega). Electrocompetent cells from mutant strains S2308Apgk and S19Apg / from B. abortus were transformed with the pBBR1-po / f construct.
  • the pgk gene was amplified using the same primer pair as above, purified from the agarose gel by the Wizard SV Gel and Clean-Up PCR System Kit (Promega), digested with restriction enzymes Bam H1 and Hind III and ligated to the plasmid. pMAL-C2. Following ligation, E. coli DH5 electrocompetent cells were transformed and DNA from recombinant colonies was subjected to restriction analysis with the enzymes Bam H1 and Hind III. Cloning was confirmed by sequencing plasmid DNA from recombinant colonies using the same primers used for pgk gene amplification.
  • this gene construct was renamed pMAL: pgk.
  • Expression of recombinant MBP-PGK protein using pMAL-c2 was performed according to the manufacturer's manual (New England Biolabs). To purify the fusion protein an amylose resin (New England Biolabs) was used.
  • mice C57BL / 6 mice were immunized with the recombinant MBP-PGK protein. Three immunizations were performed with the interval of fifteen days between each one. Seven days after each immunization, 250 ⁇ _ of retroorbital plexus blood was extracted to obtain serum containing anti-MBP-PGK antibodies. To analyze the production of specific antibodies against the recombinant protein, an indirect ELISA was performed to detect specific antibodies according to Lunde et al. (1979). For negative ELISA control sera from PBS-immunized mice were used.
  • Macrophages were obtained from bone marrow of C57BL / 6 mice. Each femur and tibia were washed with 5 ml of Hank's Balanced Salt Solution (HBSS-GIBCO). The result of the cell suspension was centrifuged and resuspended in Dulbecco's Modified Eagle's Medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS, Gibco) and 10% L929 cell conditioned medium (LCCM) as a source of M-CSF. (monocyte colony stimulating factor). Cells were distributed in 24-well plates and incubated at 37 ° C in an oven containing 5% CO 2 . After 3 days 0.1 ml LCCM was added per well.
  • DMEM Dulbecco's Modified Eagle's Medium
  • FBS fetal bovine serum
  • LCCM L929 cell conditioned medium
  • EXAMPLE 1 Analysis that the recombinant strain of Brucella bacteria is unable to synthesize the phosphoglycerate kinase enzyme
  • the wild strain, the recombinant strain and the recombinant strain supplemented with pBBRI-pg / c were grown in liquid, boiled and their contents were subjected to Western blot with anti-PGK monoclonal antibody. Wild and complemented strains showed gene expression for PGK protein, whereas recombinant strains showed no such expression (Figure 1).
  • EXAMPLE 2 Demonstration that recombinant B. abortus strain is attenuated in macrophages
  • EXAMPLE 3 Evaluation of residual virulence of S2308Apg / r and S19Apg / r mutants in different murine models
  • mice inoculated with the wild strain S2308 died between the fifth and eleventh day after infection, which was expected due to the high virulence of this strain. Eighty percent of those inoculated with the S19 vaccine strain survived during the observation period. This may be due to the residual virulence that the vaccine strain still has (Banai et al, 2002). All mice inoculated with mutant strains S2308Apgk and S19Apg and with the RB51 vaccine strain survived for four weeks after inoculation thus indicating an attenuation in the virulence of the mutant strains (Figure 3).
  • mice were sacrificed by cervical dislocation and their spleen was removed, processed and plated in serial dilution in Brucella broth (BB) medium.
  • BB Brucella broth
  • the plates were grown in an oven at 37 ° C containing 5% CO 2 .
  • the plates were counted and the number of CFU in the spleen of these animals was determined.
  • the mutant strain S2308Apg / was shown to have reduced virulence at all time points compared to wild strain S2308, and at all points this difference was significant (p ⁇ 0.05) in all mouse strains ( Figure 5).
  • mice In C57BL / 6 mice the smallest CFU difference between S2308Apgr / c and S2308 was observed in the first week (1.17 log) and the largest difference was in the sixth week (2.11 log), as can be seen in Figure 5 B. And in 129 / Sv mice the smallest CFU difference between S2308Apgk and S2308 was observed in the first week (1.45 log) and the largest difference was in the sixth week (3.24 log), as can be seen in Figure 5 C.
  • EXAMPLE 4 Evaluation of the ability of mutant strains to induce protective immune response in a murine model.
  • mice BALB / c, C57BL / 6 and 129 / Sv mice, aged 6 to 9 weeks, and IRF-1 " ' " mice, aged 6 to 12 weeks, were intraperitoneally vaccinated with 100 ⁇ of PBS containing 1x10 5 CFUs. following strains: S2308Apg / c, S19Apgk, S19 and RB51. In the control group, 100 ⁇ of PBS was inoculated. After 12 weeks the mice were challenged with 100 ⁇ of PBS containing 1x10 6 CFU of virulent strain S2308.
  • mice Fifteen days after challenge, BALB / c, C57BL / 6 and 129 / Sv mice were sacrificed by cervical dislocation and their spleen was removed, processed and plated in serial dilution in Brucella broth (BB) medium as previously described. The plates were grown in an oven at 37 ° C containing 5% CO 2 . At the end of the third day the plates were counted and the number of CFU in the spleen of these animals was determined. IRF-1 animals " '" had their survival observed daily.
  • BB Brucella broth
  • mice All vaccinated mice, both with vaccine strains and mutant strains, had lower number of Brucella in the spleen compared to control animals immunized with PBS, demonstrating that all strains were effective in activating the immune system and inducing protection (Table 1). 2) Table 2 - Protective immunity induced by S2308Apgk, S19Apgk, S19 and RB51
  • mutant strain S2308Apg / c demonstrated a level of protection similar to strain S19 (p> 0.05) and higher protection than the RB51 vaccine strain (p ⁇ 0.05).
  • mutant strain S19Apgr / demonstrated a level of protection similar to that of the RB51 vaccine strain (p> 0.05), which was shown to be less efficient than the S19 vaccine strain (p ⁇ 0.05).
  • mutant strain S2308Apg / f has been shown to induce a higher level of protection than S19 and RB51 vaccine strains (p ⁇ 0.05)
  • mutant strain S19Apg has shown a similar level of protection to that of S19 vaccine strain (p > 0.05), and both were more efficient than the RB51 vaccine strain (p ⁇ 0.05).
  • mutant strain S2308Apg / is capable of inducing an increased resistance to infection caused by B. abortus S2308 higher than S19 and RB51 vaccine strains in some murine models, with the main advantage over commercially available vaccine strains. greater attenuation in residual virulence than S19 vaccine strain with excellent protection-inducing ability.
  • the mutant strain S19Apg c increased resistance to infection to levels comparable to the RB51 vaccine strain, with the main benefit being a lower residual virulence than the S19 vaccine strain.

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Abstract

Selon l'invention, une souche recombinante S2308Δpgk de Brucella abortus est obtenue par délétion partielle ou complète du gène pgk. Cette souche atténuée induit chez les souris 129/Sv et IRF-1 KO une immunité protectrice plus importante que les souches commerciales S19 ou RB51, et une protection égale à la souche S19 chez les souris BALB/C et C57BL/6, d'où un potentiel pour être testée comme un nouveau vaccin vivant contre la brucellose. D'autres souches recombinantes de la bactérie Brucella, obtenues par délétion partielle ou complète du gène pgk, avec divers niveaux d'atténuation, sont décrites.
PCT/BR2011/000276 2010-08-04 2011-08-04 Souche recombinante de la bactérie brucella spp et vaccin vivant contre la brucellose WO2012016315A1 (fr)

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CN111394294A (zh) * 2020-04-13 2020-07-10 宁夏大学 布鲁氏菌A19 bvfA基因缺失株及其构建和应用

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KR20180042759A (ko) * 2016-10-18 2018-04-26 강원대학교산학협력단 브루셀라 어보투스 ba15 변이주 및 이를 포함하는 브루셀라병 예방용 백신 조성물
KR101868554B1 (ko) * 2016-10-18 2018-06-19 강원대학교산학협력단 브루셀라 어보투스 ba15 변이주 및 이를 포함하는 브루셀라병 예방용 백신 조성물
CN111394294A (zh) * 2020-04-13 2020-07-10 宁夏大学 布鲁氏菌A19 bvfA基因缺失株及其构建和应用
CN111394294B (zh) * 2020-04-13 2022-08-19 宁夏大学 布鲁氏菌A19 bvfA基因缺失株及其构建和应用

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