Immune response and protection conferred by mucosal immunization with BLSOmp31 using different delivery systems against Brucella ovis in rams

A. DIAZ; DANIELA A QUINTEROS; FERNANDO A. PAOLICCHI; MARIANA A. RIVERO; MARIA CLAUSSE; JUAN LLABOT; GISELLE GHERSI ; PALMA SANTIAGO; ALLEMANDI D A.; FERNANDO A. GOLDBAUM; S.M. ESTEIN

Medellin

Congreso; 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología; 2015

Frontiers

Brucella ovis causes an infectious disease in sheep characterized by epididymitis and infertility in rams and abortion in ewes. The control measures for this disease are periodical diagnosis by serological tests and/or bacteriological culture and culling of positive animals. Vaccination programs are the only viable means for the control of B. ovis in countries with a high incidence. B. melitensis Rev.1 is considered the best vaccine for the prophylaxis of ovine brucellosis but has important disadvantages associated with the development of antibodies interfering with serodiagnosis, virulence for humans and the prohibition of its use in countries considered free of B. melitensis. Consequently, there is a need for new safe and effective brucellosis vaccines to be developed. Our previous results demonstrate that a chimera of Brucella Lumazine Synthase (BLS) that contains an immunodominant epitope of Omp31 delivered as a recombinant protein (rBLSOmp31) by intramuscular (i.m.) route was immunogenic and conferred protection against B. ovis infection when rams were immunized three times. B. ovis is often transmitted from ram to ram by passive venereal transmission via ewes but the infection can be initiated when B. ovis gains entry at mucosal tissues of the gastrointestinal or respiratory tracts and the surface of the eye. Thus, intranasal (i.n.) and intraconjunctival (i.c.) immunization could be efficient for the induction of mucosal and systemic immune responses. Mucosal immunization strategies against B. ovis have not been previously investigated in sheep. In the present work we evaluated: i) the immunogenicity of the polymeric subcellular vaccine BLSOmp31 when administered by i.n. and i.c. routes using two different delivery systems: Chitosan Microspheres (ChME) and Poloxamer 407 (P407) and ii) protection conferred against B. ovis in rams. Forty Corriedale 5-month-old rams were randomly distributed in the following immunization groups (G) (n=8): (G1) BLSOmp31 in Freund?s Incomplete Adjuvant (FIA) administered by i.m. route, (G2) BLSOmp31 adsorbed on ChME administered by i.n route, (G3) BLSOmp31 formulated in P407 administered by i.c and (G4) BLSOmp31 formulated in P407 administered by i.n. routes. A fifth unvaccinated control group (G5) was included. Animals were vaccinated three times (500 g/dose, 21 days apart) and were challenged with B. ovis at day 230. Each ram received 1.09 x 109 Colony Forming Unit (CFU) by conjunctival and preputial instillation. Both serum samples and preputial, lacrimal, salival and nasal secretions were obtained at selected times during the experiment and were analyzed by indirect ELISA against BLSOmp31 to measure IgG and IgA specific antibodies. In addition, cellular immune response was evaluated in vivo by intradermal reaction to BLSOmp31 injection (day 90), and in vitro by quantification of IFN gamma in supernatant culture of blood samples stimulated with the chimera. At day 305, rams were slaughtered and necropsied. Samples of reproductive tract and extragenital organs were taken and analyzed by bacteriology. Data from serological and cellular immune responses were analyzed by ANOVA followed by Tukey post hoc tests. Chi Square Test and Fisher?s Exact test were used to analyze bacteriological data (proportion of infected or severely infected organs according to treatment). Kruskal Wallis test was used to compare numbers of infected and severely infected organs per infected ram among treatment groups. The analysis was performed using SAS v 9.3 (2011). Differences were considered significant at p<0.05. Rams immunized with BLSOmp31+AFI presented higher IgG antibody (Ab) levels than other groups in all serum and secretions samples. Immunization by mucosal routes with the two different formulations induced similar IgG antibody levels in serum. Serum IgG Ab increased after B. ovis inoculation in all groups. Induction of specific IgG Ab was lower than in serum for all groups of immunization. Nasal and preputial secretions showed higher levels of antibodies compared to lacrimal and salival secretions. Induced IgG Ab levels in nasal and preputial secretions were similar in all groups except in samples of G3. In addition, IgA specific antibodies were detected in nasal secretion for all groups of immunization. Levels of specific IgG in all groups decreased at the time of B. ovis inoculation. Significant increase of IgG Ab levels were induced in preputial secretions after challenge. Finally, parenteral and mucosal strategies of immunization stimulated similar and significant cellular immune response in vivo and in vitro. Percentages of infected and severely infected organs were significantly lower in animals immunized by parenteral and mucosal routes than unvaccinated group. Mainly, significant differences in the bacterial count in epididymides were found. However, all immunization strategies used conferred significant protection against B. ovis. Immunization with BLSOmp31 using two different delivery systems by intranasal and intraconjuntival routes of administration induced both systemic and mucosal immune response and conferred protection against B. ovis in rams.