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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 .

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.

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.

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[sup.+] CD4[sup.+] T cells, yet only znBMZ induced IFN-γ[sup.+] CD8[sup.+] T cells. While both strains induced CD4[sup.+] effector memory T cells (Tems), only znBMZ induced CD8[sup.+] Tems. Thus, these results show that the described znBM mutants are safe, able to elicit CD4[sup.+] and CD8[sup.+] T cell immunity without a boost, and highly effective, rendering them promising vaccine candidates for livestock.

The parasitic nematode Angiostrongylus cantonensis is a major cause of eosinophilic meningitis in humans, and has been documented in other incidental hosts such as birds, horses, dogs and non-human primates. It is endemic in Hawaii, and there have been sporadic reports in the southern continental United States. This parasite uses rats as definitive hosts and snails as intermediate hosts. In this study, we collected potential definitive and intermediate hosts throughout Florida to ascertain the geographic distribution in the state: Rats, environmental rat fecal samples, and snails were collected from 18 counties throughout the state. Classical diagnostics and morphological identification, along with molecular techniques were used to identify nematode species and confirm the presence of A. cantonensis. Of the 171 Rattus rattus collected, 39 (22.8%) were positive for A. cantonensis, and 6 of the 37 (16.2%) environmental rat fecal samples collected in three of the surveyed counties were also positive for this parasite by real time PCR. We examined 1,437 gastropods, which represented 32 species; 27 (1.9%) were positive for A. cantonensis from multiple sites across Florida. Three non-native gastropod species, Bradybaena similaris, Zachrysia provisoria, and Paropeas achatinaceum, and three native gastropod species, Succinea floridana, Ventridens demissus, and Zonitoides arboreus, which are newly recorded intermediate hosts for the parasite, were positive for A. cantonensis. This study indicates that A. cantonensis is established in Florida through the finding of adult and larval stages in definitive and intermediate hosts, respectively, throughout the state. The ability for this historically subtropical nematode to thrive in a more temperate climate is alarming, however as the climate changes and average temperatures rise, gastropod distributions will probably expand, leading to the spread of this parasite in more temperate areas. Through greater awareness of host species and prevalence of A. cantonensis in the United States, potential accidental infections may be avoided.

Re-emerging zoonotic pathogen Brucella spp continues to impact developing countries and persists in expanding populations of wildlife species in the US, constantly threatening re-infection of our domestic herds. Additionally, potential weaponization means the development of improved animal vaccines and an effective human vaccine remains a priority. In this study we characterize the immune response to a novel live attenuated B. melitensis strain znBM-mC using a mucosal vaccine approach in a mouse model of infection. An oral prime-intranasal boost strategy of vaccination induces protection post wt. B. melitensis pulmonary challenge comparable to that achieved with Rev 1 vaccination. Vaccination induces a local Th1-type response in the lungs as well as a robust lung B cell population. Lung T cells include memory subsets of both CD4+ and CD8+ T cells. Total B cell numbers increase significantly in the lungs of znBM-mC vaccinated mice compared with nonvaccinated controls. These B cells were primarily memory or regulatory phenotype. Both T and B cells contribute to the vaccine-induced protective response. T cells are primary producers of IFN-γ in the lung, which was essential to the early response and a primary cytokine produced pre-and post-challenge with wt B. melitensis 16M. B cells, which accumulate in the lungs post vaccination internalize Brucella antigen after challenge, with total numbers decreasing over time, a phenomenon which correlates with increased expression of apoptosis markers. We propose that in addition to the Th1-type response, B cells enhance vaccine-induced protection via a host-directed cell death. In silico epitope prediction was used to select potential MHC class I epitopes for consideration in the development of a subunit vaccine. Selected 9-mer peptide epitopes were screened in vitro using cells isolated from lungs of mice vaccinated using our znBM-mC vaccine. Four peptides were selected for testing in an in vivo model. Mucosal administration of peptides using c-di-AMP adjuvant induces a Th1 response characterized by increased IFN-γ production in bronchioalveolar lavage and cell supernatants from, lungs, head and neck lymph nodes and spleens after peptide restimulation in culture.

The parasitic nematode Angiostrongylus cantonensis is a major cause of eosinophilic meningitis in humans, and has been documented in other incidental hosts such as birds, horses, dogs and non-human primates. It is endemic in Hawaii, and there have been sporadic reports in the southern continental United States. This parasite uses rats as definitive hosts and snails as intermediate hosts. In this study, we collected potential definitive and intermediate hosts throughout Florida to ascertain the geographic distribution in the state: Rats, environmental rat fecal samples, and snails were collected from 18 counties throughout the state. Classical diagnostics and morphological identification, along with molecular techniques were used to identify nematode species and confirm the presence of A. cantonensis.