Parenteral immunization with polymeric subcellular vaccine BLSOmp31 induces serum bactericidal and opsonic antibodies against Brucella canis, and stimulates a local immune response in dogs.

5. CLAUSSE M., DÍAZ A.G., GHERSI G., GOLDBAUM F.A. , ESTEIN S.M.

Medellín

Congreso; INMUNOCOLOMBIA 2015. 11th Congress of the Latin American Association of Immunology-ALAI. 10 tColombian Congress of Allergy, Asthama and Immunology ?ACCAI.; 2015

ALAI

Parenteral immunization with polymeric subcellular vaccine BLSOmp31 induces serum bactericidal and opsonic antibodies against Brucella canis, and stimulates a local immune response in dogsMaria Clausse1, Alejandra G. Diaz1, Giselle Ghersi2, Fernando A. Goldbaum3 and SILVIA M. ESTEIN1* 1Laboratorio de Inmunología Departamento SAMP Centro de Investigación Veterinaria de Tandil (CIVETAN-CONICET) Facultad de Ciencias Veterinarias Universidad Nacional del Centro de la Pcia. de Bs. As., Argentina 2Inmunova S.A., Argentina, Argentina 3Fundación Instituto Leloir, Bs. As., Argentina, ArgentinaCanine brucellosis is the best known sexually transmitted disease in dogs. This disease is caused by Brucella canis, a naturally rough species, that is also transmittable to humans. Since diagnosis is complex and no antibiotic treatment is (a) 100% effective, a vaccine would be regarded as a desirable tool for controlling canine brucellosis.The chimeric protein (BLSOmp31) comprising the decameric enzyme lumazine synthase (BLS) decorated with a protective epitope derived from an Outer Membrane Protein of 31 kDa (Omp31) from Brucella melitensis was immunogenic and conferred protection against B. canis infection in the mouse model. In the present study, we evaluated the humoral and cellular immune response induced by the immunization with polymeric subcellular vaccine BLSOmp31 formulated in aluminum hydroxide in dogs.We used five Beagle male 5-month-old dogs that were seronegative to RSAT (Rapid Slide Agglutination Test), a screening test to detect antibodies against B. canis. Dogs were vaccinated subcutaneously three times with recombinant BLSOmp31 adsorbed to aluminum hydroxide, with an interval of 3 weeks. In addition, an annual booster was delivered. Vaccine was well tolerated without side effects and RSAT was always negative with all sera from immunized dogs throughout the study. Samples of whole blood, serum and lacrimal, nasal and preputial secretions were taken at selected times. Cellular immune response was evaluated by determination of IFN-gamma in supernatants of whole cell culture stimulated by BLSOmp31 at weeks 6 and 13. However, no significant levels of this cytokine were detected. Intradermal skin test was performed at week 13 with the recombinant protein, but no visible induration was observed at 48 or 72 hours after the injection. Thérefore, T cell responses, as measured by in vitro and in vivo experiments, were unable to be detected by the techniques used. Levels of antibodies in serum and secretions were studied by specific ELISA to analyze IgG and IgA isotypes. In addition, two tests to determine bactericidal and opsonic activity of antibodies were developed. Specific serum IgG antibodies were strongly induced after prime vaccination. Antibodies titers remained stable up to week 13 where individual significant differences were detected. At week 20 significant differences were observed in 50% of the animals, and at week 45 only one dog did not show specific IgG. After boost, IgG antibody titers reached original levels but decreased more rapidly at week 58. Therefore, as IgG1 and IgG2 are considered to be markers of Th2 and Th1 responses respectively, anti-BLSOmp31 specific antibodies of both isotypes were also measured. Immunization elicited high levels of anti-BLSOmp31 IgG1 as well as IgG2 antibodies. An important bactericidal activity of specific antibodies was demonstrated in a complement- mediated bacteriolysis assay. Serum from immunized animals showed bactericidal activity at week 10 (84%). In order to assess the serum capacity to enhance phagocytosis and killing of B. canis, an opsonocytophagic test was designed in which bacteria were mixed with serum and then were incubated with neutrophils to measure bacterial viability over time by plating at different times and counting the colony forming units. Inactivated serum of vaccinated animals significantly enhanced the neutrophils´ capacity to kill the bacteria in comparison to serum from unvaccinated dogs. To evaluate mucosal immune response, IgG and IgA isotypes were determined in salival, lacrimal and preputial secretions. Specific IgA could not be detected in any secretion. On the contrary, levels of specific IgG were found in preputial secretion of three dogs. In conclusion, humoral immune response developed against BLSOmp31 could improve B. canis elimination since it has been demonstrated that antibodies play an important role in controlling rough Brucella infection. Moreover, the presence of IgG in preputial secretion demonstrates that BLSOmp31 could elicit a local immune response in addition to the systemic immune response and could prevent bacterial colonization and subsequent infection when this bacterium enters by the venereal route. However, since formulation containing aluminum hydroxide did not stimulate a cellular response it would be interesting to evaluate different adjuvants in further studies. Nevertheless, BLSOmp31 remains a very promising candidate for a vaccine against canine brucellosis.AcknowledgementsThis work was supported by grants from the Agencia Nacional de Promoción Científica y Tecnológica (ANPCYT, Argentina). MC, AGD and GG are recipients of a postdoctoral and doctoral fellowship from the Comisión Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina). SME and FAG, and VZ are members of the Research Career of CONICET. We thank María José Del Sole and Pablo Nejamkin (U.N.C.P.B.A, Argentina) for animal care and Alberto Bujacich (Royal Canin, Argentina) for providing dog food to support this study.Conflict of InterestThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.Keywords: CHIMERIC BLSOMP31, Dogs, Parenteral Immunization, Mucosal and systemic immune response, Brucella canisConference: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología. Medellin, Colombia, 13 Oct - 16 Oct, 2015.Presentation Type: Oral PresentationTopic: Veterinary and Comparative ImmunologyCitation: Clausse M, Diaz AG, Ghersi G, Goldbaum FA and ESTEIN SM. Parenteral immunization with polymeric subcellular vaccine BLSOmp31 induces serum bactericidal and opsonic antibodies against Brucella canis, and stimulates a local immune response in dogs. Front. Immunol. Conference Abstract: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología.* Correspondence: PhD. SILVIA M. ESTEIN, , Laboratorio de Inmunología Departamento SAMP Centro de Investigación Veterinaria de Tandil (CIVETAN-CONICET) Facultad de Ciencias Veterinarias Universidad Nacional del Centro de la Pcia. de Bs. As., TANDIL, Argentina, silmares@vet.unicen.edu.ar< Back