Immune Response of Calves Vaccinated with <i>Brucella abortus</i> S19 or RB51 and Revaccinated with RB51

<div><p><i>Brucella abortus</i> S19 and RB51 strains have been successfully used to control bovine brucellosis worldwide; however, currently, most of our understanding of the protective immune response induced by vaccination comes from studies in mice. The aim of this study was to characterize and compare the immune responses induced in cattle prime-immunized with <i>B</i>. <i>abortus</i> S19 or RB51 and revaccinated with RB51. Female calves, aged 4 to 8 months, were vaccinated with either vaccine S19 (0.6–1.2 x 10¹¹ CFU) or RB51 (1.3 x 10¹⁰ CFU) on day 0, and revaccinated with RB51 (1.3 x 10¹⁰ CFU) on day 365 of the experiment. Characterization of the immune response was performed using serum and peripheral blood mononuclear cells. Blood samples were collected on days 0, 28, 210, 365, 393 and 575 post-immunization. Results showed that S19 and RB51 vaccination induced an immune response characterized by proliferation of CD4⁺ and CD8⁺ T-cells; IFN-ɣ and IL-17A production by CD4⁺ T-cells; cytotoxic CD8⁺ T-cells; IL-6 secretion; CD4⁺ and CD8⁺ memory cells; antibodies of IgG1 class; and expression of the phenotypes of activation in T-cells. However, the immune response stimulated by S19 compared to RB51 showed higher persistency of IFN-ɣ and CD4⁺ memory cells, induction of CD21⁺ memory cells and higher secretion of IL-6. After RB51 revaccination, the immune response was chiefly characterized by increase in IFN-ɣ expression, proliferation of antigen-specific CD4⁺ and CD8⁺ T-cells, cytotoxic CD8⁺ T-cells and decrease of IL-6 production in both groups. Nevertheless, a different polarization of the immune response, CD4⁺- or CD8⁺-dominant, was observed after the booster with RB51 for S19 and RB51 prime-vaccinated animals, respectively. Our results indicate that after prime vaccination both vaccine strains induce a strong and complex Th1 immune response, although after RB51 revaccination the differences between immune profiles induced by prime-vaccination become accentuated.</p></div

IFN-ɣ and IL-17A production by CD4⁺ and CD8⁺ T-cell subsets in peripheral blood mononuclear cells of S19 and RB51 prime vaccinated, and RB51 revaccinated cattle upon <i>in vitro</i> stimulation with ɣ-irradiated <i>B</i>. <i>abortus</i> 2308.

<p>Tendency (median) (a) and box plot (median, first and third quartiles) (b) charts of the results. Whiskers show the lower and upper 1.5 interquartile range. Vaccinations were indicated by arrows. Significant differences (P < 0.05) between vaccination regimens (on same day) are indicated by uppercase letters (Mann-Whitney-test), and lowercase letters indicate statistical difference between days in same group (Skillings Mack test followed by Wilcoxon signed rank test).</p

Experimental design.

<p>Forty crossbred females calves aged between 4 to 8 months were divided in two experimental groups: group S19—composed of 20 calves vaccinated with S19 vaccine strain (0.6–1.2 x 10¹¹ CFU) at day 0 of the experiment; and group RB51—composed of 20 calves vaccinated with RB51 vaccine strain (1.3 x 10¹⁰ CFU) at day 0 of the experiment. Both groups were revaccinated with RB51 (1.3 x 10¹⁰ CFU) at day 365 of the experiment. The number of animals tested in each immunological assessment (0,28, 210, 365, 393 and 575) are shown in the rectangles. The days when the vaccinations occurred are highlighted with arrows.</p

Granzyme B and perforin-expressing CD8⁺ T-cells in peripheral blood mononuclear cells of S19 and RB51 prime vaccinated, and RB51 revaccinated cattle upon <i>in vitro</i> stimulation with ɣ-irradiated <i>B</i>. <i>abortus</i> 2308.

<p>Tendency (median) (a) and box plot (median, first and third quartiles) (b) charts of the results. Whiskers show the lower and upper 1.5 interquartile range. Vaccinations were indicated by arrows. Significant differences (P < 0.05) between vaccination regimens (on same day) are indicated by uppercase letters (Mann-Whitney-test), and lowercase letters indicate statistical difference between days in same group (Skillings-Mack test followed by Wilcoxon signed rank test).</p

Gating strategies used to select specific leukocytes subpopulations.

<p>The lymphocytes were identified as R1 based on their size and granularity flow cytometric features prior to the analysis of CD8⁺, CD4⁺, CD21⁺ lymphocytes subsets identified as R2 and proliferation. Lymphocytes subpopulations expressing the memory marker (CD45RO) were quantified based on R3. The mean of fluorescence intensity of MHC class II on lymphocytes subpopulations were quantified based on R4. Percentage of lymphocytes subsets expressing intracytoplasmic cytokines (IFN-ɣ, IL-17A and IL-4) or cytotoxic markers (perforin and granzyme B) were quantified based on Q1. Percentage of lymphocytes subsets expressing FoxP3-CD25 was determined using quadrant statistics over anti-Foxp3 versus anti-CD25 marker dot plot distribution (Q2). For proliferation assay, the phenotypic analysis was carried to determine the percentage of divided cells using CFSE / anti-bovine surface marker (anti-CD4 or anti-CD8) dot plots.</p

IFN-ɣ, IL-6, IL-4 and IL-10 accumulated in cell culture supernatant of peripheral blood mononuclear cells of S19 and RB51 prime vaccinated, and RB51 revaccinated cattle upon <i>in vitro</i> stimulation with ɣ-irradiated <i>B</i>. <i>abortus</i> 2308.

<p>Tendency (median) (a) and box plot (median, first and third quartiles) (b) charts of the results. Whiskers show the lower and upper 1.5 interquartile range. Vaccinations were indicated by arrows. Significant differences (P < 0.05) between vaccination regimens (on same day) are indicated by uppercase letters (Mann-Whitney-test), and lowercase letters indicate statistical difference between days in same group (Skillings Mack test followed by Wilcoxon signed rank test).</p

Monoclonal antibodies (mAbs) against cell surface markers, intracellular cytokine, nuclear protein or immunoglobulin used in this study.

<p>^amAb purchased from AbD Serotec (Raleigh, USA).</p><p>^bmAb purchased from VMRD (Pullman, USA).</p><p>^cmAb purchased from Life Technologies (Carlsbad, USA).</p><p>^dmAb purchased from eBioscience (San Diego, USA).</p><p>^emAb purchased from R&D Systems (Minneapolis, USA).</p><p>^fmAb purchased from BD Pharmingen (San Diego, USA).</p><p>^gfluorescein isothiocyanate (FITC).</p><p>^hphycoerythrin (PE).</p><p>ⁱhorseradish peroxidase.</p><p>Monoclonal antibodies (mAbs) against cell surface markers, intracellular cytokine, nuclear protein or immunoglobulin used in this study.</p

Antibody profile of S19 and RB51 prime vaccinated, and RB51 revaccinated cattle measured by I-ELISA using S19 and RB51 whole-cell antigens.

<p>The results are expressed as mean. Data for total IgG, IgG1 and IgG2 are shown. Vaccinations were indicated by arrows. Lowercase letters indicate statistical difference between days on same group (one-way ANOVA followed by paired t-test).</p

Activation of antigen-specific IFN-γ-producing CD8+ T cell precursors in mice immunized with pIRES I or pIRES II.

<p>(A-B) Spleen cells from BALB/c mice spleen cells were stimulated with the MHC-I-restricted p24-specific peptide, and the p24-specific IFN-γ-producing CD8⁺ T cells were detected by intracellular cytokine staining (A) or ELISPOT assay (B). (C–D) Spleen cells from C57BL/6 mice were stimulated with the MHC-I-restricted E7-specific peptide, and the E7-specific IFN-γ-producing CD8⁺ T cells were detected by IFN-γ intracellular staining (C) or ELISPOT assay (D). Mice were i.m. immunized with three doses of the DNA vaccines with one week intervals between doses (100 µg/dose). The CD8⁺ T-cell responses were analyzed two weeks after the last dose. *p<0.05. Data represent the compilation of two independent experiments with four mice per immunization group (n = 8) and results expressed by each animal analyzed. pIRES is the empty vector used as immunization control.</p

CFSE proliferation analysis of CD4⁺ and CD8⁺ T-cells subsets in peripheral blood mononuclear cells of S19 and RB51 prime vaccinated, and RB51 revaccinated cattle upon <i>in vitro</i> stimulation with ɣ-irradiated <i>B</i>. <i>abortus</i> 2308.

<p>Tendency (median) (a) and box plot (median, first and third quartiles) (b) charts of the results. Whiskers show the lower and upper 1.5 interquartile range. Vaccinations were indicated by arrows. Significant differences (P < 0.05) between vaccination regimens (on same day) are indicated by uppercase letters (Mann-Whitney-test), and lowercase letters indicate statistical differences between days in same group (Skillings Mack test followed by Wilcoxon signed rank test).</p