Protection against intra-nasal challenge with virulent <i>B. abortus</i> 2308 following oral homologous prime-boost immunization of mice with multiple vaccine doses of gamma-irradiated <i>B. neotomae</i>.

a<p>Units of protection were calculated by subtracting the mean log₁₀ CFU for a vaccinated group from the mean log₁₀ CFU of the corresponding saline control group.</p><p>*Significantly different from the corresponding saline group (<i>P</i><0.05).</p><p>**Not significantly different from the corresponding saline group (<i>P</i>>0.05).</p><p>Protection against intra-nasal challenge with virulent <i>B. abortus</i> 2308 following oral homologous prime-boost immunization of mice with multiple vaccine doses of gamma-irradiated <i>B. neotomae</i>.</p

Flow cytometric analysis showing the percentage of interferon-γ secreting (A), and tumor necrosis factor-α secreting (B) CD4⁺ and CD8⁺ T cells in the spleens of BALB/c mice immunized with gamma-irradiated RB51, <i>B. neotomae</i>, RB51/<i>B. neotomae</i> and <i>B. neotomae</i>/RB51, or inoculated with saline following in vitro stimulation with media (unstimulated), gamma-irradiated <i>B. neotomae</i> and gamma-irradiated RB51.

<p>*Significantly different from the corresponding unstimulated control (<i>P</i><0.05). ^ff Significantly different from the corresponding vaccination groups with irradiated RB51 stimulation (<i>P</i><0.05).</p

Specific activity of SOD enzyme in the periplasmic extracts of <i>B. neotomae</i>, <i>B. neotomae</i>/pBB4SOD, and <i>B. abortus</i> RB51 at different growth stages.

<p>The growth curves of the bacteria are shown in the top panel. The specific activities of SOD enzyme are shown in the bottom panel. For each <i>Brucella</i>, overnight culture was used at 1∶200 dilution to inoculate fresh 100 ml media and at different time intervals, the OD₆₀₀ was measured and an aliquot of the culture was taken for preparing periplasmic extracts. With extracts of each time-point, the SOD assay was performed twice, each time in duplicates, and the results are shown as the mean ± standard deviation of specific activity (units/mg of protein). At each time point, the groups with one or two asterisks were significantly different from the <i>B. neotomae</i> group (<i>P</i><0.001). At 4 and 48 hours time-points, there were significant differences between the groups with different number of asterisks (<i>P</i><0.001).</p

Detection of SOD overexpression in <i>B. neotomae</i>/pBB4SOD and <i>B. neotomae</i>/pBB4BnSOD by SDS-PAGE.

<p>Total antigens of the indicated bacteria were prepared from different individual recombinant colonies and loaded on each lane. The gels were stained with Coomassie brilliant blue G. The lane marked MW contain molecular weight markers and the numbers at the left indicate approximate molecular masses in kilodaltons. Arrow indicates the SOD protein.</p

ELISA detection of IgG, IgM, IgA antibodies specific to <i>B. neotomae</i> LPS (A), and RB51 total antigens (B) in the intestinal secretions of mice vaccinated with gamma-irradiated RB51, <b><i>B. neotomae</i></b>, RB51/<i>B. neotomae</i> and <b><i>B. neotomae</i></b>/RB51, or inoculated with saline.

<p>Intestinal secretions were collected at 2 weeks after the last booster vaccination, were diluted 1 in 10 and assayed for the presence of LPS-specific (A) and RB51-specific (B) antibodies. Results are shown as mean ± standard deviation (<i>n</i> = 4) of absorbance at 450 nm of the color developed. *Significantly different from the corresponding saline group (<i>P</i><0.05). ^ff Significantly different from the corresponding vaccination groups (<i>P</i><0.05).</p

Oral Immunization of Mice with Gamma-Irradiated <i>Brucella neotomae</i> Induces Protection against Intraperitoneal and Intranasal Challenge with Virulent <i>B. abortus</i> 2308

<div><p><i>Brucella</i> spp. are Gram-negative, facultative intracellular coccobacilli that cause one of the most frequently encountered zoonosis worldwide. Humans naturally acquire infection through consumption of contaminated dairy and meat products and through direct exposure to aborted animal tissues and fluids. No vaccine against brucellosis is available for use in humans. In this study, we tested the ability of orally inoculated gamma-irradiated <i>B. neotomae</i> and <i>B. abortus</i> RB51 in a prime-boost immunization approach to induce antigen-specific humoral and cell mediated immunity and protection against challenge with virulent <i>B. abortus</i> 2308. Heterologous prime-boost vaccination with <i>B. abortus</i> RB51 and <i>B. neotomae</i> and homologous prime-boost vaccination of mice with <i>B. neotomae</i> led to the production of serum and mucosal antibodies specific to the smooth LPS. The elicited serum antibodies included the isotypes of IgM, IgG1, IgG2a, IgG2b and IgG3. All oral vaccination regimens induced antigen-specific CD4⁺ and CD8⁺ T cells capable of secreting IFN-γ and TNF-α. Upon intra-peritoneal challenge, mice vaccinated with <i>B. neotomae</i> showed the highest level of resistance against virulent <i>B. abortus</i> 2308 colonization in spleen and liver. Experiments with different doses of <i>B. neotomae</i> showed that all tested doses of 10⁹, 10¹⁰ and 10¹¹ CFU-equivalent conferred significant protection against the intra-peritoneal challenge. However, a dose of 10¹¹ CFU-equivalent of <i>B. neotomae</i> was required for affording protection against intranasal challenge as shown by the reduced bacterial colonization in spleens and lungs. Taken together, these results demonstrate the feasibility of using gamma-irradiated <i>B. neotomae</i> as an effective and safe oral vaccine to induce protection against respiratory and systemic infections with virulent <i>Brucella</i>.</p></div

β-Galactosidase activity in <i>B. abortus</i> virulent strain 2308 (A) and <i>B. abortus</i> vaccine strain RB51 (B) transformed with plasmids containing <i>lacZ</i> gene under the control of wildtype and the indicated mutated <i>B. neotomae sodC</i> promoters.

<p>For each promoter construct, the assays were performed using 3 separate colony cultures. Results are shown as the mean ± standard deviation of Miller units. In each panel, means with the same number of asterisks were not significantly different from each other (<i>P</i>>0.05), but there were significant differences between means with different number of asterisks (<i>P</i><0.001).</p

Evaluation of strength of <i>sodC</i> promoters from <i>B. abortus</i>, <i>B. neotomae</i>, <i>B. suis</i> biovars 2 (strain Thomsen) and 4 (strain 40).

<p>A) Nucleotide sequence features of the promoter-containing 5′ flanking region of <i>B. abortus sodC</i> gene as cloned in pBaSODpro/lacZ. The <i>sodC</i> start codon, ribosomal binding site (RBS), and 5′ ends of cDNA are indicated in bold. The asterisk indicates the site of nucleotide insertion polymorphism with <i>B. neotoame</i> and <i>B. suis</i> biovars. The two potential −10 sequences are boxed (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014112#s4" target="_blank">Discussion</a>). B) Expression levels of β-galactosidase in late log phase cultures of <i>B. neotomae</i> cultures harboring plasmids with <i>lacZ</i> gene cloned under the control of <i>sodC</i> promoters obtained from the indicated <i>Brucella</i> spp. For each promoter construct, the assays were performed using 3 separate colony cultures. Results are shown as the mean ± standard deviation of Miller units. Means with the same number of asterisks were not significantly different from each other (<i>P</i>>0.05). There were significant differences between means with different number of asterisks (<i>P</i><0.001).</p

Immunization schedule.

<p>Immunization schedule.</p

Intracellular survival and replication of <i>B. neotomae</i> and <i>B. neotomae</i>/pBB4 SOD in J774A.1 cells.

<p>Infection of J774A.1 cells was performed in triplicate as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014112#s2" target="_blank">Materials and Methods</a>, and for each time-point, results are shown as the mean ± standard deviation of log₁₀ CFU per well. At each time-point there was no significant difference between the two groups (<i>P</i>>0.05).</p