BRPI1003054B1 - Recombinant strain of bacteria brucella spp and live vaccine against brucellosis - Google Patents

Abstract

RECOMBINANT BRUCELLA BACTERIA STRAIN AND LIVE VACCINE AGAINST BRUCELLOSIS. The recombinant strain S2308(Delta)pgk of Brucella abortus S2308 is obtained by partial or complete deletion of the pgk gene. This attenuated strain induces protective immunity in 129/Sv and IRF-1 KO mice greater than commercial strains S19 or RB51, and protection equal to the S19 strain in BALB/C and C57BL/6 mice, showing potential to be tested as a novel live vaccine against brucellosis. Other recombinant strains of Brucella bacteria obtained by partial or complete deletion of the pgk gene, with varying levels of attenuation, are described.

Description

[01] The present invention pertains to the fields of immunology and molecular biology/microbiology, in particular bacteriology. More specifically, this invention describes the development of a recombinant strain of Brucella spp and its use as a vaccine for the control of Brucellosis disease in mammals.

[02] Brucellosis is a zoonosis caused by bacteria of the genus Brucella, which infect humans and domestic animals (NICOLETTI, P.L. Relationship between animal and human disease. In: Brucellosis: clinical and laboratory aspects. Young, E.J. and Corbel, M.J. CRC Press, Inc., Boca Raton, FL, p.41-51, 1989). Brucellosis is considered by the WHO as the most widespread of all zoonoses. This disease brings two concerns: health, due to its possibility of transmission to humans; and economic, due to the decrease in animal productivity, the invalidation of products and derivatives as a result of this disease, commercial restrictions, animal disposal, increase in the replacement rate of animals, death of calves and increase in the interval between calving (FRENEY, J ; RENAUD, F; HANSEN, W & BOLLET, C. Precis de bacteriologie Clinique. Paris: ESKA, p 1413-1423, 2000). In general, 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, in addition to food hygiene measures. All attempts at human vaccination have still proved ineffective and dangerous, and there is a need to develop a truly effective human vaccine to control this disease (Freney et al., 2000).

[03] Currently, vaccination against brucellosis is performed through the administration of the smooth attenuated strains S19 of B. abortus and Rev 1 of B. melitensis. There is also the rugosa strain RB51, which was recently introduced in countries such as the USA, Chile and Canada (WHO, 1998) to prevent infection by B. abortus. Mass vaccination of all females results in a reduction in economic losses, in addition to dramatically decreasing the number of human brucellosis cases (Roth F, Zinsstag J, Orkhon D, Chimed-Ochir G, Hutton G, Cosivi O, Carrin G , Otte J. Bull World Health Organ. Human health benefits from livestock vaccination for brucellosis: case study. 2003;81(12):867-76. Epub 2004 Mar 1. Erratum in: Bull World Health Organ. 2004 Jan;82( 1):76).

[04] The S19 vaccine strain, developed in the 1920s for bovine immunization, is attenuated and has a natural deletion in the EryC gene that is necessary for erythritol metabolism. This strain induces around 60% protection against infection and protects around 70% of animals for 4 to 5 pregnancies against abortion. Both the S19 strain of B. abortus and the Rev 1 strain of B. melitensis are effective in protecting against their respective pathogens, but these vaccine strains have three major disadvantages: they are pathogenic to humans, they can cause abortion when administered to pregnant females. and induce the production of antibodies in immunized animals, which interferes with the diagnosis of infected individuals or populations (CHEVILLE, N.F.; JENSEN, A.E.; HALLING, S.M.; TATUM, F.M.; MORFITT, D.C.; HENNAGER, S.G.; FRERICHS, W.M. & SCHURIG. , G. Bacterial survival, lymph node changes, and immunologic responses of cattle vaccinated with standard and mutant strains of Brucella abortus, American Journal of Veterinary Research, v. 53(10), p.1881-88, 1992;CHEVILLE, N.F.; STEVENS, M.G.; JENSEN, A.E.; TATUM, F.M. & HALLING, S.M. Immune responses and protection against infection and abortion in cattle experimentally vaccinated with mutant strains of Brucella abortus. American Journal of Veterinary Research, v. 54(10), p. 1591-97, 1993;CHEVILLE, N.F., OLSEN, S.C., JENSEN, A. E., STEVENS, A.M., FLORANCE, H.S., HOUNG, H.S., DRAZEK, E. S., WARREN, R.L., HADFIELDT. L. AND HOOVE, D. L. Bacterial persistence and immunity in goats vaccinated with a pure deletion mutant or the parental 16M strain of Brucella melitensis. Infection and Immunity. 64, p 2431-2439, 1996).

[05] The RB51 strain, on the other hand, has a modification in the structure of LPS, as it does not have the O-antigen, since it has a mutation in the wboA gene, which encodes a glycosyl-transferase involved in the polymerization of N-formyl-perosamine (MORIYÓN, I.; GRILLÓ, M.J.; MONREAL, D.; GONZÁLEZ, D.; MARÍN, C.; LÓPEZ-GONI, I.; MAINAR-JAIME, R.C.; MORENO, E. & BLASCO, J.M. Roughs in animal brucellosis: structural and genetic basis and present status. Veterinary Research, v. 35(1), p. 1-38, 2004). It is a rough strain from a smooth and virulent strain of B. abortus S2308. It was developed by serial passage in a selective medium containing sub-inhibitory concentrations of the antibiotic rifampicin (SCHURIG, G.G.; ROOP, R.M. 2ND, BAGCHI, T.; BOYLE, S.; BUHRMAN, D. & SRIRANGANATHAN, N. Biological properties of RB51; a stable rough strain of Brucella abortus, Veterinary Microbiology, v. 28(2), p. 171-88, 1991). This strain is more sensitive to complement-mediated lysis (UGALDE, J. E.; CZIBENER, C.; FELDMAN, M. F. & UGALDE, R. A. Identification and characterization of the Brucella abortus phosphoglucomutase gene: role of lipopolysaccharide in virulence and intracellular multiplication. Infection and Immunity, v. 68, p 5716-5723, 2000). The rough strain has the advantage of being 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).

[06] In order to overcome the disadvantages of currently used vaccines, several new recombinant strains were developed. WO9937783 - Live vaccine against brucellosis - describes recombinant strains of Brucella constructed by deleting the Brucella rfbU gene. This recombinant strain is attenuated and induces protective immunity similar to that induced by commercial strains.

[07] The document CN101092605 - Mutant strain of Brucella bacterin with weak poison, constructing method, and application - describes recombinant strains of Brucella constructed by deletion of the genes Bp26, wboA, omp31, and P39 or pgm of Brucella S19. These strains induce protective immunity, have the same virulence as the commercial vaccine and are unable to interfere with the diagnosis of infected individuals or populations.

[08] Document US7364745 - Development of a live, attenuated, recombinant vaccine for Brucellosis - describes recombinant strains of Brucella constructed by deleting the Brucella TspA gene. These recombinant strains are attenuated and induce protective immunity superior to that induced by commercial strains.

[09] The document CN101185756 - Brucella molecule marking and virulence deletion attenuated vaccine and preparation method - describes recombinant strains of Brucella constructed by deleting the Brucella S19 genes Bp26 and Bmp 18, making the vaccine obtained from these recombinant strains of Brucella incapable of interfere in the diagnosis of infected individuals or populations.

[10] Document CN101386833 - Recombinant bacterium of Brucella abortus and use thereof - describes a recombinant strain of Brucella constructed by deletion of the gene coding for the cold shock protein of Brucella S19. This recombinant strain is attenuated, has lower virulence than the commercial strain and is unable to interfere with the diagnosis of infected individuals or populations.

[11] Document CN101386831 - Recombinant bacterium of Brucella abortus with immunity labeling and use thereof - describes a recombinant strain of Brucella constructed by deleting the gene encoding the Brucella S19 perosamine synthetase enzyme. This recombinant strain is attenuated, has lower virulence than the commercial strain and is unable to interfere with the diagnosis of infected individuals or populations.

[12] The document US7541447 - Process for the preparation of an improved Brucella strain plasmid to develop the strain and the vaccine comprising the said strain - describes recombinant strains of Brucella constructed by deletion of the Brucella pgm gene. This recombinant strain is attenuated and induces protective immunity superior to that induced by commercial strains.

[13] Document CN101575587 - Abortus Brucella vaccine recombinant strain and application thereof in preparing vaccine - describes a recombinant strain of Brucella constructed by deleting the gene encoding the capsular polysaccharide synthesis protein and the gene encoding the carboxyl-uridine enzyme Brucella S19 phosphate decarboxylase. This recombinant strain is attenuated and unable to interfere with the diagnosis of infected individuals or populations.

[14] Document CN101575589 - Abortus Brucella vaccine strain S19 marked recombinant strain and application thereof - describes a recombinant strain of Brucella constructed by deletion of the gene encoding the Brucella S19 glycosyltransferase enzyme. This recombinant strain is attenuated and unable to interfere with the diagnosis of infected individuals or populations. The present invention involves the development of a recombinant strain of the bacterium Brucella S2308 that presents partial or complete deletion of the pgk gene, which encodes the enzyme phosphoglycerate kinase (PGK). PGK is an important enzyme of the glycolytic pathway that reversibly catalyzes the transfer of a phosphate from 1,3-bisphosphoglycerate to ADP, thus forming 3-phosphoglycerate and ATP (BERNSTEIN, B.E.; MICHELS, P.A. & HOL, W.G. Synergistic effects of substrate-induced). conformational changes in phosphoglycerate kinase activation. Nature, v. 385(6613), p. 275-8, 1997). We demonstrated that bacteria with deletion of this gene are attenuated and induce protective immunity superior to that induced by commercial strains.

[15] 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, they can cause abortion when administered to pregnant females. and they induce antibody production in immunized animals, which interferes with the diagnosis of infected individuals or populations (Cheville et al., 1992, 1993, and 1996).

[16] The RB51 rugosa strain has the advantage of being less virulent than the smooth vaccine strain, being less abortive than the other vaccine strain and not interfering 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).

[17] The recombinant Brucella abortus S2308 strain S2308Δpgk is capable of inducing protective immunity in 129/Sv and IRF-1 KO mice greater than commercial strains S19 or RB51, and equal protection to the S19 strain in BALB/c and C57BL/ mice. 6, showing potential to be used as a new live vaccine against brucellosis. Furthermore, as the mutations generated in the S19 and RB51 strains are not fully known, a major advantage of the S2308Δpgk strain is that it has a defined and known genetic alteration. Furthermore, in all models tested in vitro and in vivo, the S2308Δ pgk strain is much more attenuated than the smooth S19 strain.

BRIEF DESCRIPTION OF THE FIGURES

[18] Figure 1 - Western blot analysis of pgk expression in B. abortus S2308 (lane 1), S2308Δpgk (lane 2) and S2308Δpgk complemented with pBBR1-pgk (lane 3). The presence of a band of approximately 42 kDa in channels 1 and 3 indicates pgk expression in B. abortus strains S2308 and S2308Δpgk complemented with pBBR1-pgk.

[19] Figure 2 - Intracellular replication of S2308Δpgk and S19Δpgk mutant strains in bone marrow-derived macrophages. Adherent cells were infected with an MOI of 50 from B. abortus S2308, RB51, S2308Δpgk or S19Δpgk as described. After 2, 24, 48, 72, 120 and 168 hours, macrophages were lysed and enumerated by serial dilution. Points represent log10 CFU per well and are the mean and standard deviation of two independent experiments. Asterisks indicate statistically significant differences (P<0.05).

[20] Figure 3 - Susceptibility of IRF-1 -/- mice inoculated with the mutant strains S2308Δpgk and S19Δpgk, with the vaccine strains RB51 and S19 and with the virulent strain B. abortus S2308. Eight mice per group were infected with a dose of 1x106 CFU. Mice were monitored daily for 28 days.

[21] Figure 4 - Protection of IRF-1-/- mice against challenge with the virulent B. abortus strain S2308 after immunization with the S2308Δpgk, S19Δpgk, RB51 and S19 strains. Mice were immunized intraperitoneally with a dose of 1x105 CFU of each strain separately, except strain RB51 which were vaccinated with 1x107 CFU. Twelve weeks later they were challenged by the same route with 1x106 CFU of the virulent strain S2308. Mice were monitored daily for 30 days.

[22] Figure 5 - Persistence of the S19Δpgk mutant strain and the S19 vaccine strain in C57BL/6 (A) and 129/Sv (B) mice. Eight mice per group were infected with a dose of 1x106 CFU of each strain separately. Spleens were collected 1, 2, 3, 4 and 6 weeks after infection and the number of CFUs was determined in the macerated tissues by serial dilution and plating. Values are expressed as average CFU. The asterisks represent the statistically significant difference of the S19Δpgk group compared to the group that received the B. abortus S2308 strain (p<0.05).

DETAILED DESCRIPTION OF THE TECHNOLOGY

[23] The recombinant strain of Brucella bacteria 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 that contains a gene that confers resistance to the antibiotic kanamycin. The bacterium Brucella spp, can be selected from the group consisting of B. melitensis, B. abortus, B. suis, B. ovis, B. canis, B. neotomae, B. microti, B. inopinata, B. ceti and B. pinnipedialis. Furthermore, the live vaccine against Brucellosis developed in this technology is characterized by comprising a recombinant strain of the bacterium Brucella spp selected from the group comprising B. melitensis.DELTA.pgk, B. abortus.DELTA.pgk, B. suis.DELTA.pgk, B. ovis.DELTA.pgk, B. canis.DELTA.pgk, B. neotomae.DELTA.pgk, B. microti.DELTA.pgk, B. inopinata.DELTA.pgk, B. ceti.DELTA.pgk and B. pinnipedialis.DELTA.pgk. To this end, the methodology detailed below was used. Extraction of genomic DNA from B. abortus S2308:

[24] The extraction of genomic DNA from B. abortus S2308 was performed according to Halling et al. (1991), with some modifications. From a stock aliquot of S2308, streaking was performed on BB/Agar medium. The plates were incubated for 72 hours in an oven at 37°C containing 5% CO2. An isolated colony was inoculated into 2 ml of BB medium and incubated at 37°C under agitation at 200 rpm for 48 hours. After this time, the culture was inactivated for 1 hour at 65°C. Then it was subjected to centrifugation at 7000 rpm for 10 minutes and the pellet was resuspended in 1.5 mL of TE. Soon after total resuspension, 90 μL of SDS and 15 μL of proteinase k (20 mg/mL) were added. This mixture was incubated at 37°C for 12 to 18 hours. After this period, 300 μL of 5M NaCl and 240 μL of CTAB/NaCl were added, the mixture was homogenized and incubated at 65°C for 20 minutes. Then, an equal volume, i.e. approximately 2.145 mL, of chloroform was added and the mixture was incubated on ice and gently shaken for 15 minutes. The mixture was then centrifuged at 7000 rpm for 10 minutes. The aqueous phase was recovered and an equal volume of phenol was added thereto. The mixture was incubated on ice and gently shaken for 30 minutes, 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 subjected to centrifugation at 7000 rpm for 10 minutes. The aqueous phase was once again recovered and 0.6 mL of isopropanol was added to it so that the 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 μL of sterile milli-Q water. DNA quality and concentration were evaluated by electrophoresis in 0.8% agarose gel and analysis in a spectrophotometer at wavelengths 260 and 280 nm (UV mini 1240 UV-VIS Spectrophotometer Shimazdu). Amplification of the B. abortus pgk gene by PCR:

[25] The pgk gene (SEQ ID No 1) was amplified from the genomic DNA of B. abortus S2308, with the following primers, designed from sequences of B. abortus deposited by our group in the Gene Bank with number AF256214, using the OLIGO 4.0 program (Wojciech Rychlik). The primers used were as follows: PGKF (SEQ ID No 2): 5' GTA GGA TCC ATG ATG TTC CGC ACC CTT 3'(TM=67.64); PGKR (SEQ ID No 3): 5' GGG GGT ACC TCA CTT CTT CAA TAC ATC 3'(TM=66.12).

[26] Restriction sites inserted in the amplified fragment are highlighted in italics. For the amplification reaction, 10ng of genomic DNA was used, 1 μL of each primer (5pmoles/μL), 1 μL of 10X concentrated Taq DNA polymerase buffer (500mM Tris-HCl pH 9.0 and 1% Triton X-100 ), 0.8 μL of 25mM MgCl2, 0.25 μL of 10mM dNTPs, 1 μL of 5 U/μL Taq DNA polymerase and water q.s.p. for 10μL of reaction. The PCR reaction was performed following the following program: - First denaturation: 95° C for 3 minutes; - 30 cycles of: denaturation - 95°C for 30 seconds, annealing - 75°C for 45 seconds, extension - 72°C for 1 minute; - Final extension: 72°C for 10 minutes.

[27] After the PCR reaction, the product was subjected to electrophoresis in a 0.8% agarose gel and the fragment of interest was purified from the gel using the Wizard SV Gel and Clean-Up PCR System (Promega) kit, according to guidelines from the manufacturer. Soon after, the fragment was dosed in a 0.8% agarose gel with DNA fragments of known concentration.

[28] The amplified product and the pBluescript-KS(+) plasmid were doubly digested, in separate tubes, with restriction enzymes Bam HI and Kpn I. After digestion, the samples were subjected to electrophoresis in agarose gel 0. 8% in TAE buffer stained with ethidium bromide. The pBluescript-KS(+) vector and the 1191pb 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 a 1Kb Ladder (Invitrogen ) and DNA of known concentration.

[29] The ligation of the 1191bp fragment in the pBluescript-KS(+) vector was performed obeying the vector/fragment molar ratio of 1:3. For the ligation reaction, 2 μL of 5X binding buffer and 1.5 μL of the enzyme T4 DNA Ligase 1U/μL (Promega) were used in a final volume of 10 μL. Ligations were carried out for 16 hours at 16°C. The ligation product was renamed pBluescript-pgk. Transformation of E. coli DH5a electrocompetent cells with the pBluescript-pgk vector:

[30] E. coli DH5a competent cells were prepared using the calcium chloride technique described by Sambrook et al, 1989. For transformation with pBluescript:pgk vector, 5 μL of vector was used for 100 μL of competent cells. After electroporation, cells were centrifuged, resuspended in 100 μL of LB and plated on LB agar supplemented with 100 μg/mL of ampicillin, 50 μg/mL of X-gal and 0.5 mM of IPTG and incubated at 37°C for 16 hours. White colonies were selected and inoculated into LB medium supplemented with ampicillin (100 μg/mL). The culture was grown for 16 hours at 37°C under constant agitation.

[31] The DNA of the recombinant colonies was subjected to restriction analysis with the enzymes Bam HI and Kpn I. Cloning was confirmed by sequencing the plasmid DNA of the recombinant colonies using the same primers used in the amplification of the pgk gene. The sequences obtained were later analyzed and manually edited to eliminate ambiguities, vector regions and low quality data at the end of the sequences. Construction of pBlue-pgk-kan vector from pBluescript-pgk plasmid:

[32] The gene conferring resistance to kanamycin was obtained from plasmid pUC-4K (GE Healthcare). The Eco RI enzyme was used to extract the kanamycin gene ORF from the plasmid. The digestion reaction was performed with 1μg of the vector according to guidelines from the enzyme manufacturer, New England Biolabs. After digestion, the sample was subjected to 0.8% agarose gel electrophoresis. The fragment was removed from the gel and purified using the Wizard SV Gel and Clean-Up PCR System kit (Promega).

[33] The enzyme Eco RI was used for cloning the kanamycin gene into the pBluescript-pgk vector. The product of the link is now called pBluescript-pgk -Kan. Transformation of E. Coli DH5α electrocompetent cells with the pBluescript-pgk-kan vector:

[34] Competent E. coli DH5α cells were prepared using the calcium chloride technique described by Sambrook et al. in 1989. For the transformation with the pBluescript-pgk-Kan vector, 5 μL of vector were used for 100 μL of competent cells. Extraction of pBluescript-pgk-kan plasmid DNA from E. Coli DH5α:

[35] The pBluescript:pgk/Kan vector was extracted from E. coli DH5α by the alkaline lysis method using the Wizard mini prep kit (Promega). Plasmid quantification was estimated on 0.8% agarose gel. Electroporation of B. abortus S2308 and S19 with plasmid pBlue-pgk-kan:

[36] Electrocompetent cells from S2308 and S19 were prepared according to Halling et al. (1991), implementing some modifications. From the stock of S2308 and another of S19, striations were made in BB/agar medium, the plates were grown for 72 hours in an oven at 37°C containing 5% CO2. One colony isolated from each strain was inoculated into 2mL of BB medium and incubated at 37°C under agitation at 200 rpm for 48 hours. After this period, the saturated cultures were added to 200 mL of BB medium in a 1000 mL Erlenmeyer flask and again incubated at 37°C under agitation at 200 rpm until the OD600 was around 0.4 to 0.5. The cultures were centrifuged at 7000 rpm for 10 minutes and the pellets washed with 100 ml of sterile milli-Q water. Another centrifugation and washing step was performed. One more centrifuge 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 μL of milli-Q water plus 10% glycerol, the cells were immediately used.

[37] Electroporation was based on the protocol proposed by Drazek et al., 1995, with some modifications. The electrocompetent cells of B. abortus S2308 and S19 were placed on ice to slowly thaw. After total thawing, 5 μg of pBluescript-pgk-kan vector was added. The mixtures were placed in cooled and sterile 0.2 cm electroporation cuvettes, which were left on ice until electroporation. The electroporator (Gene-Pulser/ Pulse Coltroller, BioRad CA, USA) was set with the following parameters: 25 μF of capacitance, 2.5 KV of voltage and 400 Q of resistance. After electroporation, the cells were resuspended in 1mL of SOC medium (Invitrogen) and incubated under constant agitation at 200rpm at 37°C for 16 hours. After this period, the suspensions were plated on BB agar medium containing kanamycin. The plates were grown for 72 hours at 37°C in an oven with 5% CO 2 .

[38] Colonies isolated from the original plate were subcultured onto BB/agar plus kanamycin and BB/agar plus ampicillin with sterile tips. The plates were grown for 72 hours at 37°C in an oven with 5% CO 2 . Clones grown on both selective media were characterized as single recombinants (Kanr Ampr) and clones grown only on medium containing kanamycin were characterized as double recombinants (Kanr Amps). Kanr Amps strains were selected and colony PCR was performed with primers that amplify the kanamycin gene for initial confirmation of the mutation. After this initial confirmation, DNAs from wild-type strains were extracted to confirm the mutation using the Southern blot technique. Characterization of Kanr Amps and Kanr Ampr colonies, in which double or single homologous recombination occurred, respectively:

[39] The genetic proof that the wild-type gene was replaced by the gene interrupted by the kanamycin cassette was performed using the Southern blot technique to differentiate between single-recombinant transformants and double-recombinant transformants.

[40] Isolation of genomic DNA from recombinant clones was performed according to the methodology described above. Approximately 10 μg of the genomic DNA from each transformant was subjected to digestion with the restriction enzyme Eco RV, according to the protocol stipulated by the manufacturers. As a control, the genomic DNA of the parental strain S2308 of B. abortus and S19 digested with the same restriction enzyme was used.

[41] After digestion, samples were electrophoresed on 0.8% agarose gel and transferred to Hybond-N+ nitrocellulose membrane (GE Healthcare).

[42] Probes were constructed for the pgk gene and for the kanamycin and ampicillin resistance genes according to the following table: Table 1 - Probes used in the Southern blot.

[43] The AlkPhos Direct kit (GE Healthcare) was used to label the fragments obtained for probes. 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.

[44] The pgk gene ORF probe hybridized with an approximately 5800 bp fragment for B. abortus S2308 and an approximately 7082 bp fragment for the double recombinant clones, named S2308Δpgk. The recombinant single transformant showed a fragment of approximately 10042 bp, which corresponds to the insertion of the entire 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 genomic DNA of the transformants and did not hybridize to the genomic DNA of the wild-type S2308 strain.

[45] After confirmation that the pgk gene ORF had been replaced by the pgk gene interrupted by the kanamycin cassette in the recombinant double mutant, it was named S2308Δpgk. The same profile was observed for the transforming strain of S19, and the mutant obtained was named S19Δpgk.

Construction of the pBBR1-pgk vector:

[46] The pgk gene was amplified with the following pair of primers designed from B. abortus sequences: PGK 2F (SEQ ID NO 4): 5' GCG GGT ACC CAT GAT GTT CCG CAC CCT T 3'(TM= 63.25); PGK 2R (SEQ ID NO 5): 5' GCG GGA TCC AAC AGA TCG GAA TCA AAA CC 3'(TM=59.13).

[47] Restriction sites for the KpnI and BamHI enzymes inserted in the amplified fragment are highlighted in italics. For the amplification reaction, the genomic DNA of B. abortus was used. The PCR reaction was performed following the following program: - First denaturation: 95° C for 3 minutes; - 30 cycles of: denaturation - 95°C for 30 seconds, annealing - 61°C for 45 seconds, extension - 72°C for 1 minute; - Final extension: 72°C for 10 minutes.

[48] After the PCR reaction, the product was resolved on a 0.8% agarose gel and the fragment of interest was purified from the gel using the Wizard SV Gel and Clean-Up PCR System (Promega) kit, according to the manufacturer's instructions. . Soon after, the fragment was dosed in 0.8% agarose gel.

[49] The amplified product and plasmid pBBR1-MCS were digested with restriction enzymes Kpn I and Bam HI, purified from agarose gel using the Wizard SV Gel and Clean-Up PCR System kit (Promega) and ligated with the enzyme T4 DNA Ligase (Promega). After ligation, competent E. coli DH5 cells were transformed. The DNA of the recombinant colonies was submitted to restriction analysis with the enzymes Kpn I and Bam HI and sequencing using the same primers used in the second amplification of the pgk gene. After confirming the correct cloning of the pgk gene in the pBBR1-MCS plasmid, this gene construct was renamed pBBR1-pgk.

Transformation of recombinant strains with the pBBR1-pgk plasmid:

[50] The pBBRI-pgk vector was extracted from E. coli DH5a by the alkaline lysis method using the Wizard mini prep kit (Promega). Electrocompetent cells from the S2308Δpgk and S19Δpgk mutant strains of B. abortus were transformed with the pBBR1-pgk construct. Obtaining the MBP-PGK recombinant protein:

[51] In order to obtain anti-PGK antibodies to evaluate the production of the phosphoglycerate kinase protein in the different strains, the recombinant protein MBP-PGK was produced.

[52] The pgk gene was amplified using the same pair of primers mentioned above, purified from agarose gel using the Wizard SV Gel and Clean-Up PCR System kit (Promega), digested with restriction enzymes Bam HI and Hind III and linked to plasmid pMAL-C2. After ligation, electrocompetent E. coli DH5 cells were transformed and DNA from the recombinant colonies was subjected to restriction analysis with Bam HI and Hind III enzymes. Cloning was confirmed by sequencing the plasmid DNA of the recombinant colonies using the same primers used in the amplification of the pgk gene. After confirming the correct cloning of the pgk gene in the pMAL-C2 plasmid, this gene construct was renamed pMAL: pgk.

[53] Expression of the 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. Obtaining the anti-PGK antibody:

[54] C57BL/6 mice were immunized with the recombinant protein MBP-PGK. Three immunizations were performed with an interval of fifteen days between each one. Seven days after each immunization, 250 μL of blood was extracted from the retro-orbital plexus to obtain serum containing anti-MBP-PGK antibodies. In order to analyze the production of specific antibodies against the recombinant protein, an indirect ELISA was performed for the detection of specific antibodies according to Lunde et al. (1979). For the negative control of the ELISAs, sera from mice immunized with PBS were used. Evaluation of the growth of mutant strains in bone marrow-derived macrophages:

[55] Macrophages were obtained from the bone marrow of C57BL/6 mice. Each femur and tibia were washed with 5 ml of Hank's Balanced Salt Solution (HBSS-GIBCO). The resulting 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% CO2. After 3 days 0.1 ml of LCCM was added per well. On the 7th day of culture, the medium was renewed. On the 10th day of culture, when the cells had already differentiated into macrophages, they were infected with B. abortus. Bacterial suspensions of B. abortus S2308, RB51,2308Δpgk and S19Δpgk corresponding to the multiplicity of infection (MOI) of 50 bacteria/cell were added to the macrophages. The plates were centrifuged at 600 g for 10 min at 4 °C to synchronize the infection and the cells were incubated at 37 °C for 30 min. After this time the medium was removed and the cells washed 3 times with HBSS. After washing the cells were incubated for 90 min at 37 °C in medium containing 50 μg/ml of gentamicin (Sigma) to kill bacteria and extracellular bacteria. Then free bacteria were removed with 3 washes with HBSS and time zero was determined as described below. In the other wells, the medium was removed and replaced by DMEM plus 10% fetal bovine serum and 12.5 mg/mL gentamicin. Infected cells were washed 3 times with PBS and then lysed with 1 ml of 0.1% Triton-X 100 in ddH2O. The number of viable intracellular bacteria was determined 2h, 24h, 48h, 72h, 120h and 168h after infection. Serial dilutions of the bacterial suspension in PBS were plated in duplicate in BB with and without kanamycin (25 μg/ml). CFU was determined after 3 days of incubation in an oven at 37 °C containing 5% CO2. This experiment was done in triplicate and repeated twice.

EXAMPLE 1 - Analysis that the recombinant strain of Brucella bacteria is unable to synthesize the enzyme phosphoglycerate kinase

[56] For analysis that the recombinant strain of Brucella bacteria is incapable of synthesizing the enzyme phosphoglycerate kinase, the wild-type strain, the recombinant strain and the recombinant strain complemented with pBBR1-pgk were grown in liquid medium, boiled and their contents submitted to Western blot with anti-PGK monoclonal antibody. The wild-type and the complemented strains showed gene expression for the PGK protein, while the recombinant strain did not show such expression (figure 1).

EXAMPLE 2 - Demonstration that the recombinant strain of B. abortus is attenuated in macrophages

[57] To demonstrate that the recombinant strain of B. abortus is attenuated in macrophages, multiplication of the wild-type and recombinant strains in bone marrow-derived macrophages was evaluated. The number of viable bacteria was counted at intervals of 2, 24, 48, 120 and 168 hours after infection. To inhibit extracellular bacterial growth, gentamicin was added to the medium 30 minutes after infection. 24 hours after infection there was a difference of 2 log (P < 0.05) in the number of surviving organisms in the macrophages. The recombinant bacterium showed a lower intracellular replication rate in macrophages than the wild type at all times studied after 24 hours of incubation (figure 2), demonstrating that the recombinant bacterium has limited ability to replicate in macrophages.

[58] To determine whether bacterial growth was not altered by the absence of the pgk gene, a B. abortus S2308, S2308Δpgk, S19, and S19Δpgk bacterial growth curve in complete medium was performed. Strains S2308Δpgk and S19Δpgk were grown in the presence of kanamycin (25 mg/ml). None of the mutants showed a detectable defect in the ability to grow in complete medium.

EXAMPLE 3 - Evaluation of residual virulence of mutants S2308Δpgk and S19Δpgk in different murine models Evaluation of virulence in IRF -1 -/- mice:

[59] To determine whether the virulence of mutants S2308Δpgk and S19Δpgk had been altered, experiments were performed that verified the survival of IRF-1 -/- mice infected with Brucella. The virulence of the mutant strains S2308Δpgk and S19Δpgk was compared with the wild type S2308 of B. abortus and with the vaccine strains RB51 and S19, in groups of IRF-1-/- mice inoculated intraperitoneally with 1x106 CFU of the respective strains. During 4 weeks, the mice had their survival observed daily.

[60] All mice inoculated with the wild-type S2308 strain 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 fact may be due to the residual virulence that the vaccine strain still has (Banai et al., 2002). All mice inoculated with the mutant strains S2308Δpgk and S19Δpgk and with the vaccine strain RB51 survived for four weeks after inoculation, thus indicating an attenuation in the virulence of the mutant strains (Figure 3).

[61] The survival of IRF-/- mice infected with the mutant strains S2308Δpgk and S19Δpgk indicates that the phosphoglycerate kinase protein has some effect on the virulence of B. abortus, and the absence of this enzyme in the mutant strain caused an attenuation in its virulence . To confirm the virulence attenuation of the S2308Δpgk and S19Δpgk mutant strains, other murine models were used (figure 4). Virulence evaluation in BALB/c, C57BL/6 and 129/Sv mice:

[62] In order to evaluate the persistence of the S2308Δpgk mutant compared to the wild-type S2308 strain and the S19Δpgk mutant strain compared to the S19 vaccine strain in BALB/c, C57BL/6 and 129/Sv mice, the groups were inoculated intraperitoneally with 1x106 CFU of the wild-type S2308 strain, or the vaccine strain S19 or with the mutant strains S2308Δpgk and S19Δpgk. After 1, 2, 3, 4, 6 and 10 weeks the mice were sacrificed by cervical dislocation and their spleen was removed, processed and plated in serial dilution in Brucella broth (BB) medium. The plates were grown in an oven at 37°C containing 5% CO2. At the end of the third day, the plates were counted and the number of CFU in the spleen of these animals was determined.

[63] The S2308Δpgk mutant strain was shown to have reduced virulence at all analyzed times when compared to the wild-type S2308 strain, and at all points this difference was significant (p< 0.05) in all mouse strains (Figure 5). ).

[64] In BALB/c mice the smallest CFU difference between S2308Δpgk and S2308 was observed in the first week (0.4 log) and the largest difference was in the sixth week (3.36 log), as can be seen in Figure 5 A.

[65] In C57BL/6 mice the smallest CFU difference between S2308Δpgk 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.

[66] And in 129/Sv mice the smallest CFU difference between S2308Δpgk and S2308 was observed in the first week (1.45 log) and the biggest difference was in the sixth week (3.24 log), as can be seen in Figure 5 C.

[67] Through these different murine models, the attenuation of the virulence of the S2308Δpgk strain was confirmed as it was eliminated faster in mice than the parental strain S2308.

EXAMPLE 4 - Evaluation of the ability of mutant strains to induce a protective immune response in a murine model

[68] BALB/c, C57BL/6 and 129/Sv mice, 6 to 9 weeks of age, and IRF-1-/- mice, 6 to 12 weeks of age were vaccinated intraperitoneally with 100 μL of PBS containing 1x105 CFU of the following strains: S2308Δpgk, S19Δpgk, S19 and RB51. In the control group, 100 μL of PBS were inoculated. After 12 weeks the mice were challenged with 100 μL of PBS containing 1x106 CFU of the virulent strain S2308. 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 described above. The plates were grown in an oven at 37°C containing 5% CO2. 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.

*p <0.05, significante comparado ao grupo controle PBS. # p <0.05, significante comparado ao grupo vacinal RB51. * p <0.05, significante comparado ao grupo vacinal S19.[69] All mice vaccinated with both vaccine strains and mutant strains had lower numbers of Brucella in the spleen compared to PBS-immunized control animals, demonstrating that all strains were efficient in activating the immune system and inducing protection (Table 2). Table 2 - Protective immunity induced by S2308Δpgk, S19Δpgk, S19 and RB51.

*p <0.05, significant compared to the PBS control group. # p < 0.05, significant compared to the RB51 vaccine group. * p <0.05, significant compared to the S19 vaccine group.

[70] In BABL/c and C57BL/6 mice the mutant strain S2308Δpgk demonstrated a level of protection similar to the S19 strain (p>0.05) and superior protection than the RB51 vaccine strain (p<0.05). In these mice, the mutant strain S19Δpgk showed a level of protection similar to that of the vaccine strain RB51 (p>0.05), which was less efficient than the vaccine strain S19 (p<0.05).

[71] In the 129/Sv mouse, the mutant strain S2308Δpgk was shown to induce a level of protection superior to that of the vaccine strains S19 and RB51 (p<0.05), the mutant strain S19Δpgk demonstrated a level of protection similar to that of the vaccine strain S19 ( p>0.05), and both proved to be more efficient than the RB51 vaccine strain (p<0.05).

[72] The results showed that the mutant strain S2308Δpgk is capable of inducing an increase in resistance against the infection caused by B. abortus S2308 superior to the vaccine strains S19 and RB51 in some murine models, with the main advantage over the commercially available vaccine strains. There is a greater attenuation in residual virulence available than the S19 vaccine strain with an excellent ability to induce protection.

[73] The S19Δpgk mutant strain increased resistance against infection to levels comparable to the RB51 vaccine strain, with the main benefit being a lower residual virulence than the S19 vaccine strain.

Claims (6)

1. Recombinant strain of the bacterium Brucella spp, characterized by having the pgk gene, defined by SEQ ID N°1, modified by partial or complete deletion, being unable to synthesize the enzyme phosphoglycerate kinase. 2. Recombinant strain of the bacterium Brucella spp, according to claim 1, characterized in that the partial or complete deletion is obtained by introducing a plasmid containing the pgk gene, defined by SEQ ID No. 1, interrupted by a fragment that contains a gene conferring antibiotic resistance. 3. Recombinant strain of the bacterium Brucella spp, according to any one of claims 1 to 2, characterized in that the bacterium Brucella spp is selected from the group consisting of B. melitensis, B. abortus, B. suis, B. ovis, B. canis , B. neotomae, B. microti, B. inopinata, B. ceti and B. pinnipedialis. 4. Recombinant strain of the bacterium Brucella spp, according to any one of claims 1 to 3, characterized in that it is for preparing a vaccine for immunization or prophylaxis of mammals at risk of contracting Brucellosis. 5. Live vaccine against Brucellosis, characterized by comprising Brucella spp bacteria having the pgk gene defined by SEQ ID No. 1 modified by at least a partial deletion of such pgk gene where the Brucella spp bacterium is unable to synthesize the phosphoglycerate kinase enzyme. 6. Live Brucellosis vaccine according to claim 5, characterized in that the bacterium Brucella spp is selected from the group consisting of B. melitensis.DELTA.pgk, B. abortus.DELTA.pgk, B. suis.DELTA.pgk, B. ovis.DELTA.pgk, B. canis.DELTA.pgk, B. neotomae.DELTA.pgk, B. microti.DELTA.pgk, B. inopinata.DELTA.pgk, B. ceti.DELTA.pgk and B. pinnipedialis.DELTA.pgk.

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