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Susceptibility to brucellosis remains prevalent, even in herds vaccinated with conventional vaccines. Efforts are underway to develop an improved brucellosis vaccine, and possibly a universal vaccine, given that Brucella species are highly homologous. To this end, two B. melitensis mutants were developed, znBM-lacZ (znBMZ) and znBM-mCherry (znBM-mC), and were tested for their ability to confer systemic immunity against virulent B. melitensis challenge. To assess the extent of their attenuation, bone-marrow-derived macrophages and human TF-1 myeloid cells were infected with both mutants, and the inability to replicate within these cells was noted. Mice infected with varying doses of znBM-mC cleared the brucellae within 6–10 weeks. To test for efficacy against systemic disease, groups of mice were vaccinated once by the intraperitoneal route with either znBMZ or B. abortus S19 vaccine. Relative to the PBS-dosed mice, znBMZ vaccination greatly reduced splenic brucellae colonization by ~25,000-fold compared to 700-fold for S19-vaccinated mice. Not surprisingly, both znBMZ and S19 strains induced IFN-γ+ CD4+ T cells, yet only znBMZ induced IFN-γ+ CD8+ T cells. While both strains induced CD4+ effector memory T cells (Tems), only znBMZ induced CD8+ Tems. Thus, these results show that the described znBM mutants are safe, able to elicit CD4+ and CD8+ T cell immunity without a boost, and highly effective, rendering them promising vaccine candidates for livestock. [ABSTRACT FROM AUTHOR]

Brucellosis is a disease of livestock that is commonly asymptomatic until an abortion occurs. Disease in humans results from contact of infected livestock or consumption of contaminated milk or meat. Brucella zoonosis is primarily caused by one of three species that infect livestock, Bacillus abortus in cattle, B. melitensis in goats and sheep, and B. suis in pigs. To aid in disease prophylaxis, livestock vaccines are available, but are only 70% effective; hence, improved vaccines are needed to mitigate disease, particularly in countries where disease remains pervasive. The absence of knowing which proteins confer complete protection limits development of subunit vaccines. Instead, efforts are focused on developing new and improved live, attenuated Brucella vaccines, since these mimic attributes of wild-type Brucella, and stimulate host immune, particularly T helper 1-type responses, required for protection. In considering their development, the new mutants must address Brucella’s defense mechanisms normally active to circumvent host immune detection. Vaccination approaches should also consider mode and route of delivery since disease transmission among livestock and humans is believed to occur via the naso-oropharyngeal tissues. By arming the host’s mucosal immune defenses with resident memory T cells (TRMs) and by expanding the sources of IFN-γ, brucellae dissemination from the site of infection to systemic tissues can be prevented. In this review, points of discussion focus on understanding the various immune mechanisms involved in disease progression and which immune players are important in fighting disease. [ABSTRACT FROM AUTHOR]

Re-emerging zoonotic pathogen Brucella spp. continues to impact developing countries and persists in expanding populations of wildlife species in the US, constantly threatening infection of our domestic herds. The development of improved animal and human vaccines remains a priority. In this study, immunity to a novel live attenuated B. melitensis strain, termed znBM-mC, was characterized. An oral prime, intranasal (IN) boost strategy conferred exquisite protection against pulmonary challenge, with wild-type (wt) B. melitensis providing nearly complete protection in the lungs and spleens from brucellae colonization. Vaccination with znBM-mC showed an IFN-g+ CD8+ T-cell bias in the lungs as opposed to Rev 1-vaccinated mice showing IFN-g+ CD4+ T-cell inclination. Lung CD4+ and CD8+ effector memory T cells (TEMs) increased over 200-fold; and lung CD4+ and CD8+ resident memory T cells (TRMs) increased more than 250- and 150-fold, respectively. These T cells served as the primary producers of IFN-g in the lungs, which was essential for vaccine clearance and the predominant cytokine generated pre-and postchallenge with wt B. melitensis 16M; znBM-mC growth could not be arrested in IFN-γ−/− mice. Increases in lung TNF-a and IL-17 were also induced, with IL-17 being mostly derived from CD4+ T cells. Vaccination of CD4−/−, CD8−/−, and B6 mice with znBM-mC conferred full protection in the lungs and spleens postpulmonary challenge with virulent B. melitensis; vaccination of IL-17−/− mice resulted in the protection of the lungs, but not the spleen. These data demonstrate the efficacy of mucosal vaccine administration for the generation of protective memory T cells against wt B. melitensis. [ABSTRACT FROM AUTHOR]

Past studies with the live, double-mutant B. abortus (znBAZ) strain resulted in nearly complete protection of mice against pulmonary challenge with wild-type (wt) Brucella via a dominant CD8+ T cell response. To understand the contribution innate immune cells in priming CD8+ T cell responses, mice were nasally dosed with wt B. abortus , smooth vaccine strain 19 (S19), or znBAZ, and examined for innate immune cell activation. Flow cytometric analysis revealed that znBAZ, but not wt B. abortus nor S19 infection, induces up to a 5-fold increase in the frequency of IFN-γ-producing NK cells in mouse lungs. These NK cells express increased CXCR3 and Ki67, indicating their recruitment and proliferation subsequent to znBAZ infection. Their activation status was augmented noted by the increased NKp46 and granzyme B, but decreased NKG2A expression. Further analysis demonstrated that both lung caspase-1+ inflammatory monocytes and monocyte-derived macrophages secrete chemokines and cytokines responsible for NK cell recruitment and activation. Moreover, neutralizing IL-18, an NK cell-activating cytokine, reduced the znBAZ-induced early NK cell response. NK cell depletion also significantly impaired lung dendritic cell (DC) activation and migration to the lower respiratory lymph nodes (LRLNs). Both lung DC activation and migration to LRLNs were significantly impaired in NK cell-depleted or IFN-γ-/- mice, particularly the CD11b+ and monocytic DC subsets. Furthermore, znBAZ vaccination significantly induced CD8+ T cells, and upon in vivo NK cell depletion, CD8+ T cells were reduced 3-fold compared to isotype-treated mice. In summary, these data show that znBAZ induces lung IFN-γ+ NK cells, which plays a critical role in influencing lung DC activation, migration, and promoting protective CD8+ T cell development. [ABSTRACT FROM AUTHOR]

Re-emerging zoonotic pathogen Brucella spp. continues to impact developing countries and persists in expanding populations of wildlife species in the US, constantly threatening infection of our domestic herds. The development of improved animal and human vaccines remains a priority. In this study, immunity to a novel live attenuated B. melitensis strain, termed znBM-mC, was characterized. An oral prime, intranasal (IN) boost strategy conferred exquisite protection against pulmonary challenge, with wild-type (wt) B. melitensis providing nearly complete protection in the lungs and spleens from brucellae colonization. Vaccination with znBM-mC showed an IFN-γ + CD8 + T-cell bias in the lungs as opposed to Rev 1-vaccinated mice showing IFN-γ + CD4 + T-cell inclination. Lung CD4 + and CD8 + effector memory T cells (TEMs) increased over 200-fold; and lung CD4 + and CD8 + resident memory T cells (TRMs) increased more than 250- and 150-fold, respectively. These T cells served as the primary producers of IFN-γ in the lungs, which was essential for vaccine clearance and the predominant cytokine generated pre-and post-challenge with wt B. melitensis 16M; znBM-mC growth could not be arrested in IFN-γ -/- mice. Increases in lung TNF-α and IL-17 were also induced, with IL-17 being mostly derived from CD4 + T cells. Vaccination of CD4 -/- , CD8 -/- , and B6 mice with znBM-mC conferred full protection in the lungs and spleens post-pulmonary challenge with virulent B. melitensis; vaccination of IL-17 -/- mice resulted in the protection of the lungs, but not the spleen. These data demonstrate the efficacy of mucosal vaccine administration for the generation of protective memory T cells against wt B. melitensis ., Competing Interests: The 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., (Copyright © 2022 Goodwin, Yang, Hoffman and Pascual.)

Past studies with the live, double-mutant  B. abortus  (znBAZ) strain resulted in nearly complete protection of mice against pulmonary challenge with wild-type (wt) Brucella via a dominant CD8 + T cell response. To understand the contribution innate immune cells in priming CD8 + T cell responses, mice were nasally dosed with wt B. abortus , smooth vaccine strain 19 (S19), or znBAZ, and examined for innate immune cell activation. Flow cytometric analysis revealed that znBAZ, but not wt B. abortus nor S19 infection, induces up to a 5-fold increase in the frequency of IFN-γ-producing NK cells in mouse lungs. These NK cells express increased CXCR3 and Ki67, indicating their recruitment and proliferation subsequent to znBAZ infection. Their activation status was augmented noted by the increased NKp46 and granzyme B, but decreased NKG2A expression. Further analysis demonstrated that both lung caspase-1 + inflammatory monocytes and monocyte-derived macrophages secrete chemokines and cytokines responsible for NK cell recruitment and activation. Moreover, neutralizing IL-18, an NK cell-activating cytokine, reduced the znBAZ-induced early NK cell response. NK cell depletion also significantly impaired lung dendritic cell (DC) activation and migration to the lower respiratory lymph nodes (LRLNs). Both lung DC activation and migration to LRLNs were significantly impaired in NK cell-depleted or IFN-γ -/- mice, particularly the CD11b + and monocytic DC subsets. Furthermore, znBAZ vaccination significantly induced CD8 + T cells, and upon in vivo NK cell depletion, CD8 + T cells were reduced 3-fold compared to isotype-treated mice. In summary, these data show that znBAZ induces lung IFN-γ + NK cells, which plays a critical role in influencing lung DC activation, migration, and promoting protective CD8 +  T cell development., Competing Interests: The 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., (Copyright © 2021 Bhagyaraj, Wang, Yang, Hoffman, Akgul, Goodwin and Pascual.)