Pharmaceutical composition for prevention of brucellosis, encapsulated strain, encapsulation process and use

Abstract

Pharmaceutical composition for brucellosis prevention, encapsulated strain, encapsulation process and use. The present invention relates to a pharmaceutical composition comprising a live, attenuated mutant strain deficient in a gene encoding proteins of a specific Brucella ovis transporter, called l abcba, combined with a mechanism for the gradual and continuous release of this strain into the host, plus pharmaceutically acceptable excipients and adjuvants. For gradual release, the dabcba strain of Brucella ovis was encapsulated in alginate. The invention also relates to the encapsulated áabcba strain of Brucella ovis, the encapsulation process and the use of the described pharmaceutical composition in the prevention of brucellosis in sheep. The encapsulated B. ovis dabcba strain confers protection and elicits a cellular response in sheep experimentally infected with B. ovis.

Description

1 / 27 Pharmaceutical composition for the prevention of brucellosis, encapsulated strain, encapsulation process and use. [1] This Addition Certificate arises from the Invention Patent application PI 1105977-0, filed on December 22, 2011, entitled “ATTENUATED BRUCELLA OVIS STRAIN, VACCINE COMPOSITION AND USE”. [2] The present invention relates to a pharmaceutical composition comprising a live, attenuated mutant strain deficient in a gene encoding proteins of a specific ABC transporter of Brucella ovis, called AabcBA, coupled with a mechanism for the gradual and continuous release of this strain into the host, plus pharmaceutically acceptable excipients and adjuvants. For gradual release, the AabcBA strain of Brucella ovis was encapsulated in alginate. The invention also relates to the encapsulated AabcBA strain of Brucella ovis, the encapsulation process, and the use of the described pharmaceutical composition in the prevention of brucellosis in sheep. The encapsulated B. ovis AabcBA strain confers protection and elicits a cellular response in sheep experimentally infected with B. ovis. [3] Brucellosis is an infectious disease caused by bacteria of the genus Brucella. These bacteria affect domestic and wild animals and humans (FRANCO, ✜. P.; MULDER, ✜.; GILMAN, ✜. RH. Human brucellosis. Lancet Infect. Diseases, v. 7, n. 12, p. 775-786, 2007), causing large economic losses (SANTOS, R. L.; MARTINS, ✜. TM.; BORGES, ✜. AM.; PAIXÃO, ✜. TA. Economic losses due to bovine brucellosis in Brazil. Pesq. Vet. Bras., v. 33, p. 759-764, 2013). [4] Brucella ovis is a Gram-negative, non-encapsulated, non-motile bacillus belonging to the family α2-Protobacteriaceae (GARRITY, GM Bergey's Manual of Systematic Bacteriology, 2nd ed. Springer Press, New York, 2001), which does not cause harm to human health, but can cause chronic infection in sheep (XAVIER, M. N.; SILVA, T. M. A.; COSTA, E.A.; et al. Development and evaluation of a species-specific PCR assay for the detection of Brucella ovis infection in rams. Vet. Microbiol., v. 145, p. 158-164, 2010; SILVA, A.P.C.; MACEDO, A.A.; COSTA, L.F.; et al. Brucella ovis lacking a species-specific putative ATP-binding cassette transporter is attenuated but immunogenic in rams). The main clinical manifestations of the disease are Epididymitis in males and abortions in females (BLASCO, JM Brucella ovis. In: NIELSEN, K., 2 / 27 DUNCAN, JR (Eds), Brucellosis Animal. CRC Press, Boca Raton, FL, p. 351-378, 1990; THOEN, CO; ENRIGHT, F.; CHEVILLE, NF BRUCELLA. IN: GYLES CL, THOEN CO, eds. Pathogenesis of bacterial infections in animals. Ames: Iowa State University Press, pp. 236-247, 1993). [5] Due to the serious problems brought about by brucellosis to domestic animals and producers, such as infertility and subfertility in males or the birth of weak lambs, much has been done to prevent this disease through the implementation of various vaccination strategies (NICOLETTI, P. Vaccination against Brucella. Adv. Biotechnol. Processes, v. 13; p. 147-168, 1990). [6] Despite attempts to develop effective and safe vaccines (LECLERQ, S.; HARMS, JS; ROSINHA, GM; et al. Induction of a Th1-type of immune response but not protective immunity by intramuscular DNA immunization with Brucella abortus GroEL heat-shock gene. J. Med. Microbiol., v. 51, p. 20-26, 2002), there is no specific, available, and safe vaccine against B. ovis. The ideal vaccine against B. ovis should prevent infection and clinical signs or, at least, reduce the risk of infection and transmission (DÍAZ, AG; CLAUSSE, M.; PAOLICCHI, MAF; et al. Immune response and serum bactericidal activity against Brucella ovis elicited using a short immunization schedule with the polymeric antigen BLOmp31 in rams. Vet. Immunol. [7] Rev 1, a live attenuated vaccine strain of B. melitensis, is the most commonly used vaccine against B. melitensis, also providing protection against B. ovis (RIDLER, AL; WEST, DM Control of Brucella ovis infection in sheep. Vet. Clin. North Am. Food Anim. Pract., v. 27, n. 1, p. 61-66, 2011). However, Rev 1 has pathogenic potential, being able to infect humans and cause abortion in small ruminants, in addition to being resistant to streptomycin (BLASCO, JM A review of the use of B. melitensis Rev 1 vaccine in adult sheep and goats. Prev. Vet. Med., v. 31, p. 275-83, 1997; GRILLÓ, MJ; BOSSERAY, N.; BLASCO, JM In vitro markers and biological activity in mice of seed lot strains and commercial Brucella melitensis Rev.1 and Brucella abortus B19 vaccines. Biologicals, v. 28, p. PLOMMET, M.; VERGER, JM (Eds.), Brucella melitensis.Martinus Niijhoff, Dordrecht, pp. 247-251, 1985). Furthermore, in countries. 3 / 27 such as Brazil, where B. melitensis is considered exotic, or in countries where this bacterium has already been eradicated, the marketing and use of this vaccine are prohibited (ROBINSON, A. Guidelines for coordinated human and animal brucellosis surveillance In FAO ANIMAL PRODUCTION AND HEALTH PAPER 156, 2003). [8] Mice have been used as an infection model for the study of the pathogenesis of human and animal brucellosis (ENRIGHT FM The pathogenesis and pathobiology of Brucella infection in domestic animals. Chapter 12. In Brucellosis animal (K. Nielsen & JR Duncan, eds). CRC Press, Boca Raton, Florida, 301320, 1990; FICHT, TA Discovery of Brucella virulence mechanisms using mutation analysis KO, J.;. 16, p. 65-78, 2003; SILVA, TMA; COSTA, EA; PAIXAO, TA; et al. Laboratory animal models for brucellosis research. J. Biomed. Biotechnol., p. 1-9, 2011; VIEIRA, ALS; SILVA, TMA; MOL. JPS; et al. MyD88 and TLR9 are required for early control of Brucella ovis infection in mice. Res.MA; COSTA, EA; et al. The type IV secretion system encodes virB is critical for the establishment and persistence of Brucella ovis infection in mice. Vet. Microbiol., v. 159, p. 130-140, 2012), since there are many difficulties for experimental studies with production animals. Thus, alternatives have been taken to evaluate animal infection and vaccine responses to B. ovis in new experimental models (JIMENEZ DE BAGUÉS, MP; ELZER, PH; BLASCO, JM; et al. Protective immunity to Brucella ovis in BALB / c mice following recovery from primary infection or immunization with subcellular vaccines. Infect. Immun., v. 62, p. 632-638, 1994; CASSAS 2007). [9] In patent document PI 1105977-0, entitled “ATTENUATED BRUCELLA OVIS STRAIN, VACCINE COMPOSITION AND USE”, it is demonstrated that the deletion of a gene encoding proteins of the ABC transporter system of B. ovis leads to 4 / 27 attenuation of this strain in mice (SILVA T. M. A.; PAIXÃO, TA; COSTA, EA; et al. Putative ATP-binding cassette transporter is essential for Brucella ovis pathogenesis in mice. Infect. Immun., v. 79, p. 1706-1717, 2011). This attenuation suggests that this strain may serve as a vaccine sample against B. ovis infection in sheep.

[10] Research conducted over the past 100 years has demonstrated that the best vaccination strategy against brucellosis is the use of live, attenuated microorganisms, as these offer better protection against the disease (VERSHILOVA, PA. The use of live vaccine for vaccination of human beings against brucellosis in the USSR. Bull World Health Organ., v. 24, p. 85-89, 1961; DAVIS, DS; ELZER, PH. Brucella vaccines in wildlife. 2002). Furthermore, these characteristics, combined with a mechanism for the gradual and continuous release of these agents into the host (microencapsulation), increase the safety and efficacy of the vaccine, since the antigen will persist longer in the body, leading to a more elaborate immune response (FICHT, T.; KAHL-MCDONAGH, M.M.; ARENAS-GAMBOA, AM; RICE-FICHT, AC.).Brucellosis: The case for live, attenuated vaccine. Vaccine, vol. 27, p. 40-43, 2009).

[11] Currently, an alternative to enhance the efficiency of vaccines is the administration of immunogens in compartments or capsules. The encapsulation of immunogens is a tool used to improve the host's immune response through the stimulation of antigenic uptake, in addition to the fact that the capsules, which are erodible, can also function as a reservoir of antigens, releasing them slowly in order to prolong their availability in the body, thus developing a more elaborate immune response (RICE-FICHT, A.; KAHL-MCDONAGH, M. M.; FICHT, T.; Arenas-gamboa, A. Polymeric particles in vaccine delivery. Curr. Opin. Microbiol., v. 13, p. 106-112, 2010).

[12] Synthetic and natural materials such as polycaprolactone and alginate are widely used for encapsulating immunogens. Research has shown that the use of polycaprolactone as a microparticle, a synthetic polymer, 5 / 27 biodegradable, biocompatible and low cost, associated with components of the outer membrane of B. ovis, conferred protection and humoral response to sheep similar to the response conferred by Rev-1 (MUNOZ, PM; ESTEVAN M.; MARIN, CM; DE MIGUEL MJ; GRILLÓ, MJ; BARBERÁN, M.; IRACHE, JM; BLASCO, JM; GAMAZO, C. Brucella outer membrane complex-loaded microparticles as a vaccine against Brucella ovis in rams.

[13] Alginate is a natural, non-toxic, biocompatible biocompound found throughout the world (KARNITZ, O.; GURGEL, LVA; GIL, LF Removal of Ca2+ and Mg2+ from aqueous single metal solutions by mercerized cellulose and mercerized sugar-cane bagasse grafted with EDTA dianhydride (EDTAD). Carboh. Pol., v. 79, n.1, p. 184-191, 2010) and low cost (KRASAEKOOPT, W.; BHANDARI, B.; DEETH, H. Evaluation of encapsulation techniques of probiotics for yogurt. Inter. Dairy J., v. 13, n. 1, p. 3-13, 2003). This compound has been widely used to encapsulate live attenuated strains of Brucella abortus and B. melitensis, resulting in increased immunogenicity and protection in mice (ARENAS-GAMBOA, AM; FICHT, TA; KAHL-MCDONAGH, M. M.; RICE-FICHT, AC Immunization with a single dose of a microencapsulated Brucella melitensis mutant enhances protection against wild-type challenge).M.; et al. The Brucella abortus S19 kvjbR live candidate is safer than S19 and confers protection against wild-type vaccine challenges in BALB / c mice when delivered in a sustained-release vehicle. Infect. Immun., v. 77, p. 877-884, 2009) and in deer (ARENAS-GAMBOA, M.; FICHT, TA; DAVIS, DS; et al. Enhanced immune response of red deer {Cervus elaphus) to live RB51 vaccine strain using composite microspheres. J. Wild. Dis., v. 45, p. 165-173, 2009a).

[14] Patent documents and scientific articles describing vaccines for protection against brucellosis, comprising attenuated strains or encapsulated antigens, were found in the state of the art.

[15] US patent document 2014 / 248354, entitled “Controlled release vaccines and methods for treating Brucella diseases and disorders”, describes pharmaceutical compositions comprising attenuated strains of Brucella melitensis and Brucella abortus, for use as vaccines. These compositions 6 / 27 They possess an in vivo controlled-release system for the vaccine, which consists of encapsulating the attenuated strain in alginate. The ability of this vaccine to induce a protective immune response was evaluated in mice.

[16] US patent document 2006153868, entitled “Process for the preparation of an improved Brucella strain plasmid to develop the strain and the vaccine comprising the said strain”, describes an attenuated strain of Brucella abortus, obtained by deleting the pgm gene that encodes the phosphoglucomutase protein, the process for obtaining this attenuated strain and its use as a vaccine against brucellosis.

[17] Patent document WO2012 / 016315, entitled “A recombinant strain of the bacterium Brucella spp. and live vaccine against brucellosis”, describes the development of an attenuated mutant strain of Brucella abortus obtained by partial or complete deletion of the pgk gene, which encodes phosphoglycerate kinase, and that this strain induces protective immunity in mice.

[18] The scientific article entitled “Oral vaccination with microencapsulated strain 19 vaccine confers enhanced protection against Brucella abortus strain 2308 challenge in red deer (Cervus elaphus elaphus)” describes an oral or subcutaneous vaccine suitable for wildlife, comprising the attenuated S19 strain of Brucella encapsulated in alginate and vitelline protein B. Oral use of this vaccine in red deer resulted in significantly reduced bacterial load when compared with controls after Brucella challenge (ARENASGAMBOA, AM; FICHT, T. A.; DAVIS, DS; ELZER, PH; Kahl-McDonagh, M.; Wong-Gonzalez, A.; Rice-Ficht, AC Oral vaccination with microencapsulated 19 vaccine confers enhanced protection against Brucella abortus strain 2308 challenge in red deer (Cervus elaphus elaphus).

[19] However, there is no description in the state of the art of a live attenuated encapsulated vaccine that has proven immunogenic potential in sheep against Brucella ovis infection. Nor is the △abcBA mutant strain of B. ovis encapsulated in alginate and its use as a vaccine described.

[20] In view of the need for a safe and available vaccine in Brazil against B. ovis, the present technology proposes pharmaceutical compositions 7 / 27 comprising a live, attenuated mutant strain deficient in a gene encoding proteins of a B. ovis-specific ABC transporter (AabcBA) encapsulated with alginate, whose protective and immunogenic potential has been demonstrated in sheep. BRIEF DESCRIPTION OF THE FIGURES

[21] In Figure 1, the frequency of sheep serologically positive by agar gel immunodiffusion (AGID) experimentally infected with the virulent strain or Brucella ovis AabcBA is shown over twenty-four weeks of infection. Six sheep were used per group.

[22] In Figure 2, the frequency of detection of the virulent strain of Brucella ovis or Brucella ovis AabcBA by bacterial isolation and PCR in biological samples (y-axis) from experimentally infected sheep over twenty-four weeks is shown. (A) Bacteriological culture of urine samples, (B) Bacteriological culture of semen samples; (C) PCR of urine samples, (D) PCR of semen samples.

[23] Figure 3 shows the percentage of lymphocyte proliferation in sheep experimentally infected with the virulent strain of Brucella ovis AabcBA. The columns represent the mean of six unstimulated control sheep (CN); or six sheep inoculated with the virulent strain of B. ovis (B. ovis); or six sheep inoculated with B. ovis AabcBA (AabcBA B. ovis); or phytohemagglutinin (PHA). The data represent mean and standard error. Means were compared using Tukey's test. Asterisks indicate a statistically significant difference between the groups (* p <0.05; ** p <0.01).

[24] Figure 4 shows the graphical representation of leukocyte immunophenotyping in peripheral blood of sheep not infected or infected with the virulent strain or Brucella ovis AabcBA at the first (1), fourth (4) or eleventh (11) weeks after infection. (A) CD4+ T lymphocytes, (B) CD8+ T lymphocytes, (C) B lymphocytes, (D) monocytes and (E) Tg / Δ lymphocytes. Data represent mean and standard error. Means were compared using the Kruskal-Wallis test. Asterisks indicate statistically significant differences between groups (** p <0.01). 8 / 27

[25] Figure 5 shows the frequency (%) of Brucella ovis detection by bacterial culture (A) or PCR (B) in organs of sheep experimentally infected with the virulent strain or B. ovis AabcBA 24 weeks after infection. Six sheep per group.

[26] Figure 6 is an optical microscopy image of the alginate capsule (5x, bar at 500pm) (A); and fluorescence microscopy of an alginate capsule containing Brucella ovis AabcBA expressing mCherry (5x, bar at 500pm) (B).

[27] Figure 7 shows the logarithm of Brucella ovis colony-forming units isolated in the spleen (A) and liver (B) of male BALB / c mice immunized or not with B. ovis AabcBA and then challenged with the virulent strain of B. ovis. Each bar represents the mean of 5 (negative control), 5 (PBS SC), 5 (PBS IP), 10 {AabcBA B. ovis SC) and 10 {AabcBA B. ovis IP) mice with standard deviation. Raw data underwent logarithmic transformation before ANOVA and means were compared using Tukey's test. Statistical differences are indicated by asterisks (* p < 0.05; *** p < 0.001).

[28] Figure 8 shows the logarithm of Brucella ovis colony-forming units recovered in the spleen (A) and liver (B) of male BALB / c mice immunized three times with B. ovis AabcBA and unimmunized mice challenged with the virulent strain of B. ovis. Each bar represents the mean of 10 unimmunized mice and 10 immunized mice with standard deviation. Raw data underwent logarithmic transformation before ANOVA and means were compared using Tukey's test. Statistical differences are indicated by asterisks (*** p < 0.001).

[29] Figure 9 shows the logarithm of Brucella ovis colony-forming units isolated in the spleen (A) and liver (B) of BALB / ce mice and in the spleen (C) and liver (D) of C57BL6 male mice inoculated with PBS and alginate and immunized with encapsulated or non-encapsulated B. ovis AabcBA and then challenged with the virulent strain of B. ovis. Each bar represents the mean of 5 (non-immunized), 5 (alginate), 5 (B. ovis AabcBA) and 5 (B. ovis AabcBA encapsulated) mice with standard deviation. Raw data underwent logarithmic transformation before ANOVA and means were compared using Tukey's test. 9 / 27 Statistical differences are indicated by asterisks (* p < 0.05; ** p < 0.01; *** p < 0.001).

[30] Figure 10 shows images of unimmunized or immunized male BALB / ce C57BL6 mice with encapsulated Brucella ovis AabcBA and challenged with the virulent strain of B. ovis. Splenomegaly (arrowhead) and microgranulomas (arrows) in the liver of unimmunized BALB / c (A) and C57BL6 (C) mice. Spleen without size alteration in BALB / c (B) and C57BL6 (D) mice immunized with encapsulated B. ovis AabcBA and absence of microgranulomas in the liver.

[31] Figure 11 shows microscopic changes in unimmunized male C57BL6 mice or those immunized with encapsulated Brucella ovis AabcBA and challenged with the virulent strain of B. ovis. Microgranulomas in the liver of an unimmunized C57BL6 mouse (arrow) associated with an extensive area of ​​necrosis, H.E. 20x (A) in contrast to a discrete microgranuloma in the liver of a C57BL6 mouse immunized with the encapsulated mutant strain, H.E. 20x, bar at 100 µm (B). Numerous neutrophils with an extensive area of ​​necrosis in the spleen of an unimmunized C57BL6 mouse, HE, 40x (C) and rare neutrophils in the spleen of a C57BL6 mouse immunized with encapsulated B. ovis AabcBA, HE, 40x, bar at 50 µm (D).

[32] Figure 12 shows the median spleen and liver lesion score (combined inflammatory lesion score) of non-immunized, alginate-inoculated, and encapsulated and non-encapsulated Brucella ovis AabcBA male BALB / c (A, B) and C57BL6 (C, D) mice. Each bar represents the mean of 5 (non-immunized), 5 (alginate), 5 (B. ovis AabcBA) and 5 (encapsulated B. ovis AabcBA) mice with standard deviation.

[33] Figure 13 shows the frequency of non-immunized sheep, sheep immunized with encapsulated Brucella ovis AabcBA, or sheep that were not serologically positive by agar gel immunodiffusion (AGID) before and after challenge with B. ovis. The arrows indicate the week of immunization and challenge, and the numbers on the x-axis represent the week number after immunization and challenge. Statistical differences between the groups (10 sheep per group) are indicated by asterisks (**p<0.01). 10 / 27

[34] In Figure 14, the frequency of detection of the virulent strain of Brucella ovis is represented by bacteriology and PCR in biological samples (y-axis) before and after challenge with B. ovis. The arrows indicate the week of immunization (with B. ovis AabcBA encapsulated with alginate or not) and challenge. (A) bacteriological culture of semen samples, (B) bacteriological culture of urine samples, (C) PCR of semen samples, (D) PCR of urine samples.

[35] In Figure 15, the frequency of detection of the virulent strain of Brucella ovis is represented by bacteriology (A) and PCR (B) in tissues of sheep not immunized and immunized with encapsulated or non-encapsulated B. ovis AabcBA.

[36] Figure 16 shows images of macroscopic changes in non-immunized sheep after challenge. (A) Epididymal asymmetry, (B) granuloma between the visceral and parietal layers of the tunica vaginalis adjacent to the epididymal tail (arrow), (C) fibrous adhesion between the testis and the epididymal head (arrow), and (D) fibrinous adhesion in the tunica vaginalis (arrow).

[37] Figure 17 shows images of microscopic changes in sheep that were not immunized and immunized with the mutant strain of Brucella ovis encapsulated after challenge. (A) Intense neutrophilic epididymitis associated with cystic epithelial degeneration (arrow), with positive immunostaining for B. ovis (smaller quadrant, 100X) in the epididymal tail of a non-immunized sheep; (B) Normal epididymal tail of a sheep immunized with encapsulated B. ovis AabcBA after challenge; (C) Moderate neutrophilic and lymphohistiocytic ampullitis in a non-immunized sheep; (D) Normal ampulla of a sheep immunized with encapsulated B. ovis AabcBA after challenge, H.E., 40X, bar at 50 µm.

[38] Figure 18 shows the frequency of detection of non-immunized and immunized sheep with encapsulated or non-encapsulated Brucella ovis AabcBA with inflammatory changes in reproductive organs, spleen, liver and iliac lymph nodes.

[39] Figure 19 shows the percentage of lymphocyte proliferation in non-immunized sheep, immunized with the Brucella ovis AabcBA strain encapsulated or not with alginate post-immunization (A) and post-challenge (B). The columns represent the average of 10 non-immunized sheep; or 10 sheep immunized with B. ovis AabcBA, or 10 sheep immunized with encapsulated B. ovis AabcBA. The data represent mean and standard deviation. The means were compared by 11 / 27 Kruskal-Wallis test. Asterisks indicate a statistically significant difference between the groups (* p <0.05; “ p <0.01). DETAILED DESCRIPTION OF THE TECHNOLOGY

[40] The present invention relates to a pharmaceutical composition comprising a live, attenuated mutant strain deficient in a gene encoding proteins of a specific ABC transporter of Brucella ovis, called AabcBA, combined with a mechanism for the gradual and continuous release of this strain into the host, plus pharmaceutically acceptable excipients and adjuvants. More precisely, the technology relates to a pharmaceutical composition comprising the AabcBA strain of Brucella ovis encapsulated in alginate plus pharmaceutically acceptable excipients and adjuvants. The invention also relates to the encapsulated AabcBA strain of Brucella ovis, the encapsulation process, and the use of the described pharmaceutical composition in the prevention of brucellosis in sheep. The encapsulated B. ovis AabcBA strain confers protection and elicits a cellular response in sheep experimentally infected with B. ovis.

[41] The attenuated strain of Brucella ovis is characterized by comprising a mutation in genes that encode for the ABC transporter. The mutant strain of Brucella ovis (TMS3) was constructed by directed mutagenesis, through the deletion of two ORFs (open reading frames) that encode proteins that make up an ABC-type transporter system. These ORFs are located on a genetic island specific to the species of Brucella ovis that causes disease in sheep, when compared to other classic species of Brucella that infect domestic animals.

[42] Encapsulation of the strain in alginate can be carried out using the process described below: (a) addition of cells (0.5 x 1011 to 2 x 1011 CFU of B. ovis) to a sodium alginate solution (1%); (b) Slow dripping of the mixture obtained in (a) into a calcium chloride (CaCl2) solution (0.5%); 12 / 27 (c) homogenization of the capsules formed in (b) for 15 minutes followed by washing in MOPS buffer solution (4-morpholinepropanesulfonic acid); (d) adding the capsules obtained in (c) to a suspension of Poly-L lysine (0.5%) (15 minutes) under agitation; (e) washing the capsules obtained in (d) in MOPS buffer solution; (f) addition of the particles obtained in (e) to an alginate solution (0.03%) (5 minutes); (g) washing the particles obtained in (f) in a MOPS buffer solution.

[43] The dripping described in step (b) can be performed with the aid of an insulin syringe. The insulin syringe can preferably be 0.2 mL with a 0.23 x 4 mm needle.

[44] The homogenization described in step “c” can be carried out using a benchtop shaker.

[45] The invention also describes the use of the above-described composition as a live vaccine against brucellosis. This pharmaceutical composition is useful in preventing Brucella ovis infections in sheep. The vaccine is preferably administered subcutaneously.

[46] Rams immunized with this vaccine and challenged with Brucella did not eliminate the virulent strain in urine or semen and, when analyzing tissues of the reproductive system, spleen, liver and iliac lymph node of these rams, the virulent strain was also not found, unlike the non-immunized rams. Furthermore, there were no alterations during the clinical examination and no lesions were observed in the rams during necropsy and microscopy. According to the results of immunization with the alginate-encapsulated mutant strain B. ovis habcBA, it can be stated that this is immunogenic and confers protection to rams against experimental infection by B. ovis.

[47] The present invention can be better understood, without limitation, by means of the following examples. Example 1: Infection kinetics of the Brucella ovis AabcBA strain in sheep

[48] ​​ABC transport systems in bacteria are associated with nutrient uptake and export of toxins and antibiotics, having an important function in 13 / 27 Gene expression of Brucella spp. ABC transporter proteins have pathogenic potential during infection in the host. With the deletion of the gene of an ABC transporter system in B. ovis, attenuation of this strain can be observed in mice, as already described in patent document PI 1105977-0. Based on these results, this attenuated mutant strain may have high immunogenic and protective potential in sheep.

[49] Thus, it was considered important and necessary to investigate the infection kinetics of this attenuated mutant strain in sheep in order to subsequently evaluate its use as a vaccine strain in this species.

[50] Twelve one-year-old, uncastrated, crossbred rams were used. The rams were fed hay and sheep feed with 18% protein twice a day. They had water and special mineral salt for sheep available in the paddock ad libitum. Six rams were challenged with 2 mL of a suspension containing 1.2 x 109 CFU / mL of B. ovis strain ATCC25840 intrapreputially and 50 pL in both conjunctival sacs of a suspension containing 1.2 x 1010 CFU of the same strain, and the other six rams were challenged with 2 mL of a suspension containing 1.2 x 109 CFU / mL of the B. ovis strain AabcBA intrapreputially and 50 pL in both conjunctival sacs of a suspension containing 1.2 x 1010 CFU of the same strain. Following infection, weekly blood, semen, and urine samples were collected for serological evaluation, PCR, bacteriology, and semen smear testing for six months.For semen collection, the animals were conditioned prior to inoculation by introducing a female into the herd weekly. After inoculation, the animals were separated and kept in different paddocks (six animals in each paddock), with different handlers.

[51] After six months of challenge, the animals were deeply sedated with 2% xylazine hydrochloride (0.1 mg / kg intravenously, Copazine, Schering-Plough Coopers, Brazil), anesthetized with sodium thiopental (intravenously, Cristalia, Brazil) and then euthanized by electrocution. After euthanasia, fragments of the tail, head and body of the epididymis, testis, vesicular gland, bulbourethral gland, ampulla, glans, prepuce, iliac lymph nodes, spleen and liver were collected and used for bacterial isolation, histopathology, DNA extraction and PCR techniques. 14 / 27

[53] This experiment was approved by the Animal Experimentation Ethics Committee (CETEA / UFMG, protocol no. 204 / 2012).

[54] Results demonstrated that a sheep infected with the virulent strain of B. ovis became serologically positive by agar gel immunodiffusion (AGID) in the first week post-infection, while all remaining sheep infected with the virulent strain or B. ovis AabcBA seroconverted only in the fourth week after infection. Both groups of sheep remained seropositive until the sixteenth week post-infection. At 24 weeks post-infection, 50% (3 / 6) of sheep inoculated with the virulent strain of B. ovis and 33.33% (2 / 6) of sheep infected with B. ovis AabcBA remained seropositive (Figure 1).

[55] To evaluate the persistence of the virulent strain and B. ovis AabcBA in experimentally infected rams, urine and semen samples were subjected to bacteriological culture and polymerase chain reaction (PCR). B. ovis AabcBA was not detected in urine or semen samples by either bacteriological culture or PCR at any time interval during the course of infection (i.e., during 24 weeks post-infection). On the other hand, the virulent strain of B. ovis was detected in urine by bacteriological culture from the fifth week post-infection, and 33.33% (2 / 6) of the rams continued to shed the organism in their urine until the last week post-infection (Figure 2A). Detection of B. ovis in semen by bacteriology began from the fourth week post-infection (Figure 2B). When detection was assessed by PCR, the virulent strain of B. ovis was detected in urine samples starting in the fourth week after infection.Similar to the results obtained with urine bacteriological culture, PCR also indicated that 33.33% (2 / 6) of the rams continued to eliminate the virulent strain of B. ovis in the urine for 24 weeks after infection (Figure 2C). Detection of B. ovis in semen by PCR began from the fifth week after infection (Figure 2D).

[56] To evaluate lymphocyte immunoproliferation, a cell proliferation assay was performed. There was a significant increase in lymphocyte proliferation when lymphocytes from sheep challenged with the virulent strain or B. ovis AabcBA at the tenth week after infection were compared with lymphocytes obtained from the unstimulated control (Figure 3). 15 / 27

[57] In order to evaluate the blood leukocyte profile of sheep experimentally infected with the virulent strain and B. ovis AabcBA, immunophenotyping was performed. The percentage of CD4+ T lymphocytes increased in both infected groups (virulent strain and B. ovis AabcBA) when compared with the uninfected control in the first week post-infection. In the fourth and eleventh weeks after infection, the percentage of CD4+ T lymphocytes did not differ significantly when infected sheep were compared with uninfected controls (Figure 4A). The percentage of CD8+ T lymphocytes in peripheral blood was significantly higher in both infected groups (virulent strain and AabcBA) in the fourth and eleventh weeks after infection, when compared with uninfected controls (Figure 4B).There was no statistically significant difference in CD8+ T cell levels in the first week post-infection, neither in the group infected with the virulent strain nor in the AabcBA group compared to the control. The percentage of B lymphocytes, monocytes, and γ / Δ lymphocytes were statistically similar among all groups throughout the course of infection (Figures 4C, 4D, and 4E). No difference was observed between animals infected with the mutant and virulent strains throughout the experiment.

[58] To assess the detection of virulent and B. ovis AabcBA strains in reproductive system tissues, spleen, liver, and iliac lymph node of rams, bacterial culture and PCR were performed. The B. ovis AabcBA strain was not detected in any of the tissue samples collected for bacteriological culture (Figure 5A). This strain was detected by PCR in the iliac lymph node of only one ram (Figure 5B). In contrast, all rams infected with the virulent strain of B. ovis had evidence of infection, either by bacteriological culture or PCR. With the exception of the spleen, all other organs of rams infected with the virulent strain of B. ovis had varying frequencies of positivity by bacteriological culture. Of the most affected tissues, 83.33% (5 / 6) of sheep infected with the virulent strain of B. ovis had infection in the tail and head of the epididymis, while 66.66% (4 / 6) of sheep infected with the virulent strain of B.ovis samples from the vesicular glands were bacteriologically positive (Figure 5A). Similar results were obtained by PCR, but additionally, the virulent strain of B. ovis was detected in 66.66% (4 / 6) of the vesicular glands by PCR (Figure 5B). 16 / 27

[59] Before infection, no inflammatory cells were observed in any of the semen samples from the 12 rams. At 4 weeks post-infection, 16.61% (3 / 6) of the animals infected with B. ovis had a slight amount of inflammatory cells in the ejaculate (predominantly neutrophils, with some other leukocytes). At 8 weeks post-infection, 50% (3 / 6) of these animals had a moderate accumulation of neutrophils in the ejaculate. At 12 weeks post-infection, 66.66% (4 / 6) of the rams had inflammatory cells in the ejaculate, which was recurrent at 16, 20, and 24 weeks post-infection. In contrast, only one animal infected with the AabcBA strain had a slight amount of inflammatory cells at 12 weeks post-infection, which was absent at all other infection times (Table 1). Table 1 - Inflammatory cells in the semen of rams experimentally infected with Brucella ovis and AabcBA at different infection times. Semi-quantitative evaluation in semen: (-) absent, (+) slight, (++) moderate. Weeks post-infection Animal Group ............................................................................. _ 1 - - + + + + - - 2 - - - + + - - - 3 + + ++ ++ 4-4- B. ovis 4 - - - - + - + - 5 - - - * - - - - 6 - - - - - - + - 7 : - / . - - - - - - i 8 - - - * B. ovis 9 : - . - . - - - - - . _ 1 AabcBA 10 - - - 11 - - • . - + - 12 - - - · : - ' • - . -

[60] It can be stated, then, that B. ovis AabcBA, which lacks a species-specific functional ABC transporter, triggers a serological response profile 17 / 27 which is similar to that of the virulent strain. Furthermore, the mutant is capable of initiating a significant host cellular response as evidenced by a lymphocyte proliferation assay. In addition, the mutant strain is not shed in semen or urine, which is a desirable characteristic for a vaccine strain. Taken together, these results support the hypothesis that this mutant has high potential for testing as a vaccine strain. Example 2: Encapsulation of the AabcBA strain of Brucella ovis in alginate.

[61] For encapsulation of the B. ovis AabcBA mutant strain, a solution containing 1011 CFU of B. ovis was added to a 1% sodium alginate solution (Sigma-Aldrich). This mixture was placed in an insulin syringe (0.2 mL) with a 0.23 x 4 mm needle and slowly dripped into 10 mL of a 0.5% calcium chloride (CaCl2) solution. After dripping, capsules were formed, then homogenized for 15 minutes and washed twice in MOPS buffer solution (4-morpholinepropanesulfonic acid). The capsules were then placed in a 0.5% Poly-L lysine suspension (Sigma-Aldrich) for 15 minutes under agitation and then washed in MOPS buffer solution. The particles were added to a 0.03% alginate solution for 5 minutes and then washed in a MOPS buffer solution. The particles were inoculated subcutaneously at a final dose of 10⁸ CFU / mouse.To evaluate the CFU count in the capsules, they were placed in a depolymerizing solution (a solution containing sodium citrate, sodium chloride, and MOPS) for 10 minutes under agitation, and then these capsules were diluted in PBS and plated on TSA.

[62] Particle size was assessed using optical and scanning electron microscopy. The effectiveness of bacterial encapsulation was evaluated by encapsulating B. ovis AabcBA expressing mCherry using fluorescence microscopy (Leica DM 4000 B). Alginate capsules were evaluated by optical microscopy, and B. ovis AabcBA expressing mCherry was encapsulated with alginate and evaluated by fluorescence microscopy. It was observed that empty capsules or those containing B. ovis have similar size and shape. These capsules range from 300 to 800 µm in diameter (Figure 6A). Numerous colonies 18 / 27 red and fluorescent B. ovis AabcBA bacteria expressing mCherry were observed inside alginate capsules, confirming that B. ovis was efficiently encapsulated (Figure 6B). Example 3: Evaluation of the B. ovis kabcBA strain encapsulated in alginate in mice.

[63] To test the vaccine, thirty-five male BALB / c mice, aged six to eight weeks, were initially used. One group of ten mice was immunized once intraperitoneally and another ten mice were immunized subcutaneously with 100 pL of a suspension containing 108 CFU of the B. ovis AabcBA strain. Another ten mice were inoculated with sterile PBS (5 subcutaneously and 5 intraperitoneally). Six weeks after immunization, 30 mice were challenged intraperitoneally with 100 pL of a suspension containing 106 CFU of the virulent B. ovis strain (ATCC25840). The remaining five mice were inoculated intraperitoneally with PBS only and used as a negative control.After two weeks of challenge, the mice were euthanized with prior anesthesia of xylazine hydrochloride (2%, 0.1 mg / kg, intraperitoneal, Copazine, Schering-Plough Coopers, Brazil) and ketamine hydrochloride (1%, 35 mg / kg, intraperitoneal) followed by cervical dislocation.

[64] The results showed that there was no statistically significant difference in the recovery of bacteria in the spleen of the different groups evaluated (Figure 7A), however, in the liver of mice vaccinated both subcutaneously (p < 0.001) and intraperitoneally (p < 0.05) there was a significant reduction in the number of CFU recovered compared to their respective controls (Figure 7B).

[65] Since there was no difference in CFU recovery when comparing the different immunization routes used in this experiment, the subcutaneous route was used, as it is the most commonly used for vaccinations.

[66] To verify the increase in protection in a murine model of the B. ovis kabcBA strain after repeated vaccination, three subcutaneous immunizations with the mutant strain B. ovis AabcBA were performed on the animals at an interval of one 19 / 27 weeks between each vaccination. For this, 20 male BALB / c mice, aged 6-8 weeks, were used. One group of 10 mice was immunized subcutaneously with 100 pL of a suspension containing 10⁸ CFU of the B. ovis AabcBA strain, three times, with a one-week interval between immunizations. The remaining 10 mice were inoculated with sterile PBS three times, with a one-week interval between immunizations. Six weeks after immunization, all mice were challenged intraperitoneally with 100 pL of a 10⁶ CFU suspension of a virulent B. ovis strain (ATCC25840). After two weeks of challenge, the mice were euthanized with prior anesthesia of xylazine hydrochloride (2%, 0.1 mg / kg, intraperitoneal, Copazine, Schering-Plough Coopers, Brazil) and ketamine hydrochloride (1%, 35 mg / kg, intraperitoneal) followed by cervical dislocation.This experiment was approved by the Ethics Committee on Animal Experimentation (CETEA / UFMG, protocol no. 204 / 2012). No significant difference was observed in the number of bacteria recovered in the spleen of immunized mice when compared to non-immunized mice (Figure 8A), however, there was a statistically significant difference (p < 0.001) when this comparison was made in the liver (Figure 8B).

[67] To evaluate whether there is increased protection when the B. ovis AabcBA strain is encapsulated with alginate, 20 male BALB / c mice (a strain susceptible to B. ovis infection) and 20 C57BL6 mice (a strain resistant to B. ovis infection) (Fernandes et al., 1996; Murphy et al., 2001), aged between six and eight weeks, were used, divided into four groups (for each strain) containing five mice in each group: one group inoculated with PBS, a second group immunized only with sterile alginate capsules, a third group immunized only with the mutant strain of B. ovis (100 pL of a suspension containing 108 CFU) and a fourth group immunized with the mutant strain of B. ovis encapsulated with sterile alginate (100 pL of a suspension containing 108 CFU), all by subcutaneous route. After 8 weeks of immunization, all groups were challenged with the virulent strain of B. ovis (100 pL of a suspension containing 106 CFU) via intraperitoneal injection.Two weeks after the challenge, the mice were euthanized following anesthesia with xylazine hydrochloride (2%, 0.1 mg / kg, intraperitoneal) and ketamine hydrochloride (1%, 35 mg / kg, intraperitoneal). 20 / 27 TA followed by cervical displacement. This experiment was approved by the Ethics Committee on Animal Experimentation (CETEA / UFMG, protocol no. 395 / 2013).

[68] It was observed that the bacterial load was reduced (approximately by one log) in the spleen of BALB / c mice immunized with encapsulated B. ovis AabcBA when compared to the other groups (Figure 9A). In the liver, there was a reduction in the number of bacteria recovered in mice immunized with encapsulated B. ovis AabcBA when compared to non-immunized mice or those inoculated with sterile alginate (Figure 9B). There was also a significant reduction in the bacterial load recovered from the spleen (Figure 9C) and liver (Figure 9D) of C57BL6 mice immunized with the encapsulated mutant strain when compared to the other groups. Analysis of the protection index of the different presentation forms of the B. ovis AabcBA vaccine strain showed that encapsulation with alginate increased the strain's protection potential (Table 2). Table 2 - Protection conferred by Brucella ovis AabcBA encapsulated with or without alginate against B. ovis in BALB / ce C57BL6 mice. Vaccine strain Route / Dose Number of immunizations Mouse Challenge Protection index B. ovis AabcBA IP / 1x10⁸ 1 BALB / c B. ovis (IP / 1X10⁶) - 0.21 (±0.12) B. ovis AabcBA SC / 1x10⁸ 1 BALB / c B. ovis (IP / 1x10⁶) -0.17 (±0.18) B. ovis AabcBA SC / 1x10⁸ 3 BALB / c B. ovis (IP / 1x10⁶) -0.34 (±0.32) B. ovis AabcBA encapsulated with alginate SC / 1x10⁸ 1 BALB / c B. ovis (IP / 1 x10⁶) 0.54 (± 0.15)* B. ovis AabcBA encapsulated with alginate SC / 1x10⁸ 1 C57BL6 B. ovis (IP / 1x106) 1.01 (±0.12)* Statistically significant difference (p < 0.05) compared to the other groups. 21 / 27

[69] In macroscopic examination, there was no significant change in either the size or weight of the spleen of BALB / c (Figures 10A and 10B) and C57BL6 (Figures 10C and 10D) mice immunized with the encapsulated B. ovis AabcBA strain compared to the other groups, more visually evident in the latter lineage.

[70] Furthermore, in the liver of mice not immunized with the encapsulated strain, it was possible to observe circular areas of approximately 0.3 cm, whitish and firm, characterized as microgranulomas (Figures 10A and 10C), which were not seen in the liver of mice immunized with this encapsulated strain (Figures 10B and 10D).

[71] Histopathology showed that, in the liver of non-immunized mice inoculated with alginate and immunized with the non-encapsulated mutant strain, there were numerous microgranulomas in the parenchyma characterized by multifocal random lymphohistiocytic and neutrophilic inflammatory infiltrate associated with multifocal necrosis (Figure 11 A), moderate multifocal and moderate accumulation of fibrin with neutrophils and macrophages in the intima of arterioles of the portal space. In the liver of mice immunized with the encapsulated mutant strain, there was a significant decrease in the number of microgranulomas (Figure 11B). In the white pulp and marginal zone of the spleen of mice not immunized with the encapsulated strain, there was a large number of neutrophils with an area of ​​necrosis, characterizing an intense multifocal neutrophilic splenitis associated with necrosis (Figure 11C), unlike those immunized with the encapsulated strain, in which there are rare neutrophils (Figure 11 D).

[72] Scores were assigned to the inflammatory lesions observed in the spleen and liver of the different groups of BALB / ce C57BL6 mice. The mean score of the lesions observed in the spleen and liver of BALB / c mice immunized with the encapsulated strain was significantly lower (p < 0.01) when compared to the lesions in non-immunized mice inoculated with alginate (Figures 12A and 12B). In the comparison of the lesion score in the spleen of C57BL6 mice, the results were similar, however the P-value was < 0.05 (Figure 12C). In the comparison of the lesion score in the liver, the results were similar to those of the spleen and liver lesions of the BALB / c mice (Figure 12D). 22 / 27

[73] The absence of macroscopic lesions in the spleen and liver, a decrease in the number of microgranulomas in the liver and a decrease in neutrophilic inflammatory infiltrate in the spleen are indicative of good protection, thus, these positive results are encouraging for the use of the B. ovis àabcBA strain as a vaccine in sheep. Example 4: Evaluation of the B. ovis AabcBA strain, encapsulated or not in alginate, in sheep.

[74] To evaluate the B. ovis kabcBA strain, encapsulated or not in alginate, as a vaccine in sheep, thirty one-year-old crossbred, uncastrated sheep, negative by serology and urine PCR for B. ovis, were used. The sheep were fed hay and sheep feed with 18% protein twice a day. They had water and special mineral salt for sheep available in the paddock ad libitum. Ten sheep were inoculated subcutaneously with 2 mL of PBS, another 10 animals were immunized subcutaneously with 2 mL of a suspension containing 1 x 109 CFU of the B. ovis AabcBA strain, and the other 10 were immunized with the mutant strain of B. ovis AabcBA encapsulated with alginate, subcutaneously, with the same volume and concentration as the previous group. The sheep were kept in separate paddocks (ten sheep in each paddock), without direct contact and with different handlers.After a two-month period, the animals were challenged with 2 mL of a solution containing 1.2 x 10⁹ CFU / mL of B. ovis strain ATCC25840 via intrapreputial injection and 50 pL in both conjunctival sacs of a solution containing 1.2 x 10¹⁰ CFU of the same strain. Preputial and conjunctival inoculations were performed simultaneously, so that each animal received a challenge of 3.6 x 10⁹ CFU of B. ovis. The animals underwent biweekly collections, two months before and two months after the challenge, of serum samples for AGID serological testing, blood for immunoproliferation and immunophenotyping, as well as semen samples for smear, bacterial isolation and PCR reaction, and urine for bacterial isolation and PCR reaction. At the end of the experiment, the sheep were deeply sedated with xylazine hydrochloride (2%, 0.1 mg / kg, intravenously, Copazine, Schering-Plough Coopers, Brazil), and anesthetized with an overdose of sodium thiopental (intravenously). 23 / 27 (Cristalia, Brazil) and then euthanized by electrocution. After euthanasia, fragments of the tail, head and body of the epididymis, testicle, seminal vesicle, bulbourethral gland, ampulla, glans, prepuce, iliac lymph nodes, spleen and liver were collected and used for bacterial isolation, histopathology, DNA extraction and PCR techniques. This experiment was approved by the Animal Experimentation Ethics Committee (CETEA / UFMG, protocol no. 204 / 2012).

[75] The results showed that ninety percent (9 / 10) of the sheep immunized with encapsulated B. ovis AabcBA and 70% (7 / 10) of the sheep immunized only with the mutant strain became serologically positive by IDGA two weeks after immunization.

[76] Both groups of sheep remained seropositive until the fourth fortnight post-immunization. As expected, non-immunized sheep were not positive for B. ovis before the challenge. In the first two weeks after the challenge, there is a significant decrease in seropositive animals in both the group immunized with the encapsulated mutant strain (3 / 10; p < 0.001) and the group immunized only with the non-encapsulated mutant strain (6 / 10; p < 0.05). In the following two weeks, the number of seropositive sheep increased in both groups: those immunized with the encapsulated mutant strain increased to 90% and those immunized only with the mutant strain increased to 60%. In the eighth week after the challenge, the percentage of seropositive sheep immunized with the encapsulated strain is 90%, while the percentage of non-immunized sheep decreased to 70%, as did those immunized with the non-encapsulated strain (Figure 13).

[77] To evaluate the protection induced by the encapsulated or non-encapsulated B.ovis AabcBA strain, urine and semen samples were collected and subjected to bacteriological culture and PCR. During the four fortnights after immunization, there was no bacterial growth in either the semen or urine of the B. ovis AabcBA vaccine sample in the immunized rams. After the challenge, none of the rams immunized with the encapsulated or non-encapsulated mutant strain shed B. ovis in the semen or urine. Only the unvaccinated rams shed B. ovis during four fortnights (Figures 14A and 14B). 24 / 27

[78] B. ovis DNA was not detected in the semen or urine of rams vaccinated with the mutant strain encapsulated or not with alginate, being detected only in semen samples from non-immunized rams (Figures 14C and 14D).

[79] To assess the detection of B. ovis DNA in tissues of the reproductive system, spleen, liver and iliac lymph node of sheep, bacterial culture and PCR were performed.

[80] The virulent strain of B. ovis was not detected by bacteriological culture in any of the tissue samples collected from animals immunized with the encapsulated and non-encapsulated mutant strain B. ovis AabcBA, being detected only in tissues from non-immunized sheep. Of all the tissues evaluated from non-immunized sheep, B. ovis was not detected only in the spleen (Figure 15A).

[81] When B. ovis DNA was detected by PCR in the tissues of sheep immunized with the encapsulated or non-encapsulated mutant strain, no tissue was positive. In 80% (8 / 10) of the non-immunized sheep, the head and body of the epididymis, testis, vesicular gland, prepuce and spleen were positive by PCR (Figure 15B).

[82] No inflammatory cells were observed in any of the semen samples from all rams before the challenge, even after immunization of the groups with encapsulated or non-encapsulated B. ovis AabcBA strain. From the first fortnight post-challenge, almost all non-immunized rams (8 / 10) presented inflammatory cells in the ejaculate (predominantly neutrophils, with some lymphocytes) and the quantity of these cells varied from slight to intense. Four rams immunized with the non-encapsulated mutant strain did not eliminate inflammatory cells in the semen and only one ram vaccinated with the encapsulated mutant strain, at a single time, eliminated a moderate quantity of lymphocytes and plasma cells in the semen (Table 3). Table 3 - Inflammatory cells in the semen of rams not immunized and immunized with encapsulated or non-encapsulated Brucella ovis AabcBA and challenged with the virulent strain of B. ovis after immunization and after the challenge. Semi-quantitative evaluation in semen (-) absent, (+) slight, (++) moderate, (+++) intense. Animal Group Weeks post-vaccination Weeks after the challenge 25 / 27 2 4 6 8 2 4 6 8 1 - - - - +++ +++ + ++ 2 - - - - - + ++ ++ 3 - - - - +++ ++ + ++ 4 - - - - +++ +++ +++ +++ No 5 — - - - + + - - immunized 6 - - - - + + + + 7 - - - - + + + + 8 - - - - + ++ - - 9 - - - - + +++ + ++ 10 - - - - - - - +++ 11 - ..... ++ λ - 12 — . - _ ·· - ;S '1: . λ- · ·.:-» ... „13 c - .. .. . . - - - -ih: +++ + ++ • .-7.2... .>;~·* > *:·«= = · 14 - i.·' - ·- +-·? · b: 15 : - . ......... -· · A:· > . +..uu - · ovis AabcBA 16 - - - 'ii - ...... - 17 *· - . ' - - Ϊ 18 - - - + ++ + ;.· +. - - - - - B. ovis 25 - - - - - ÂabcBA 26 __ - - - - - encapsulated 27 - - - • - - - 28 - - - - - - - - 29 - - - - - - - - 30 - - - - - ++ - - Semi-quantitative evaluation in semen: (-) absent, (+) slight, (++) moderate, (+++) intense. 26 / 27

[83] After the first two weeks post-challenge, during clinical examination, some non-immunized rams showed significant and unilateral enlargement of the epididymal tail (mainly left), leading to asymmetry (Figure 16A) and pain on palpation. In the following two weeks, it was possible to feel during scrotal palpation testicular flaccidity (probably due to testicular degeneration) and nodular structures, approximately 2x2 cm, firm in the left epididymal tail of some non-immunized rams (granulomas). Immunized rams did not show any alterations during clinical examination throughout the experiment.

[84] During necropsy, no lesions were observed in rams immunized with the encapsulated B. ovis AabcBA mutant strain or in those immunized with the non-encapsulated mutant strain. However, in non-immunized animals, rounded structures, approximately 3x2x1 cm, reddish-yellow, firm, could be observed in the visceral layer of the tunica vaginalis near the epididymis tail (Figure 16B), fibrous adhesions between the portion of the tunica albuginea that covers the testicle and the portion that covers the epididymis head (Figure 16C), as well as fibrinous adhesion in the tunica albuginea (Figure 16D), in addition to edema in the tunica vaginalis.

[85] Microscopically, moderate or intense neutrophilic and lymphohistiocytic inflammatory infiltrate was observed, mainly in the tail (20%) (Figure 17A) and head (20%) of the epididymis, ampulla (20%) (Figure 17C), gallbladder (30%), iliac lymph nodes (70%), liver (10%), spleen (60%), and tunica vaginalis (20%) of non-immunized sheep. Minimal histopathological changes were observed in animals immunized with the non-encapsulated mutant strain, such as discrete lymphocytic inflammatory infiltrate in the gallbladder and urethral bulb (Figure 18). Tissue fragments from sheep immunized with the alginate-encapsulated mutant strain did not show significant histological changes (Figures 17B and 17D).

[86] The proliferation assay was performed to evaluate the cellular immune response of the encapsulated or non-encapsulated B. ovis AabcBA strain.

[87] A significant increase in the percentage of lymphocytes was observed in both sheep immunized with encapsulated B. ovis AabcBA and those immunized 27 / 27 with the non-encapsulated mutant strain compared to non-immunized sheep after immunization (p < 0.5) (Figure 19A) and after the challenge (p < 0.01) (Figure 19B).

[88] According to the results presented, it can be stated that the mutant strain 8. ovis AabcBA encapsulated with alginate is immunogenic and confers protection to sheep against experimental infection by B. ovis.

Claims

1 / 1 CLAIMS 1. Vaccine composition for the prevention and treatment of Brucellosis comprising, as the active component, the live attenuated strain of Brucella ovis ΔabcBA, which is characterized by a mutation of the gene encoding the ABC transporter (TMS3), carried out by deleting the portions before and after two ORFs (open reading frames), BOVA0500 and BOVA0501, which encode proteins that make up an ABC-type transporter system, characterized by the strain being encapsulated in alginate, plus pharmaceutically acceptable excipients and adjuvants.

2. Attenuated strain of Brucella ovis ΔabcBA containing a mutation in the gene encoding the ABC transporter (TMS3), achieved through the deletion of the portions preceding and following two ORFs (open reading frames), BOVA0500 and BOVA0501, which encode proteins that comprise an ABC-type transporter system, characterized by being encapsulated in alginate. Petition 870260065274, dated 02 / 07 / 2026, p. 7 / 7

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