Parenteral Vaccination with a Live Brucella melitensis Mutant Protects against Wild-Type B. melitensis 16M Challenge

Abstract

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.

Keywords: Brucella; IFN-γ; T cells; TNF-α; vaccine.

Figure 1

Two recombinant strains, ∆znuA ∆norD B. melitensis-lacZ (znBMZ) and ∆znuA ∆norD B. melitensis-mCherry (znBM-mC), show reduced growth in (A) primary mouse bone-marrow-derived macrophages (BMDMs) and (B) human TF-1 myeloid cells. (A) Wild-type (wt) B. melitensis 16M (BM 16M), B. melitensis Rev 1 vaccine, znBMZ, and znBM-mC were used to infect BMDMs (1 × 10⁴/well) at a bacteria-to-macrophage ratio of 10:1. Primary BMDMs were derived from BALB/c mouse femurs and recovered BMDMs were found to be >96% CD11b⁺ F4/80⁺. After a 3-h incubation followed by a 30 min treatment with 50 μg/mL gentamicin to rid extracellular brucellae, infected BMDMs were incubated in fresh medium for 0, 24, 48, or 72 h. Infected macrophages were water-lysed, and supernatants were diluted for CFU enumeration on potato infusion agar (PIA). The level of initial infection was the same for all Brucella strains (t = 0 h). The results show that both znBMZ and znBM-mC were unable to achieve the level of colonization as the wt BM 16M strain or B. melitensis Rev 1 vaccine. (B) Human TF-1 myeloid cells (1 × 10⁴/well) were infected 10:1 with wt BM 16M, B. melitensis Rev 1 vaccine, znBMZ, or znBM-mC, and evaluated for colonization as described above. The results showed that both znBMZ and znBM-mC were unable to achieve the same level of colonization as wt BM 16M or Rev 1. Values are the means of triplicate wells ± SEM: *** p ≤ 0.002; differences in brucellae colonization by the same strain over time vs. t = 0; ^### p ≤ 0.001; differences in brucellae colonization vs. wt BM 16M at the same time point; and ^§§ p < 0.01, ^§§§ p < 0.001 differences in brucellae colonization vs. BM Rev 1 vaccine.

Figure 2

The Brucella mutant, znBM-mC, is attenuated since it is effectively cleared from mice following intraperitoneal (IP) delivery. BALB/c mice (4–7/group/time point) were IP vaccinated with 1 × 10⁶ CFUs of B. abortus S19 or with the indicated dose of znBM-mC (10⁵, 10⁶, or 10⁷ CFUs). At 3, 6, and 10 weeks (wks) after vaccination, individual spleens were assessed for (A) brucellae colonization and (B) splenic weights. Values are the mean CFUs or weights from individual mice ± SEM; *** p < 0.001, ** p ≤ 0.02, * p < 0.05 differences vs. S19 colonization at the specific time point; ^## p = 0.018 between mice dosed with 10⁵ or 10⁶ CFUs of znBM-mC at 6 wks post-infection.

Figure 3

A single IP vaccination with znBMZ protects mice against wt Brucella colonization. Groups of BALB/c mice were IP vaccinated with sterile PBS vehicle (n = 4), 10⁷ CFUs S19 (n = 7), or 10⁷ CFUs znBMZ (n = 6) on day 0. On day 42, all mice were challenged IP with 5 × 10⁴ CFUs wt BM 16M. Spleens from individual mice were harvested and evaluated (A) for brucellae colonization on PIA plates with X-gal and (B) for individual weights. Values are the mean CFUs or weights from individual mice ± SEM; *** p < 0.001, ** p ≤ 0.01, differences vs. PBS-treated mice; ^††† p < 0.001 vs. S19-vaccinated mice.

Figure 4

IP vaccination with znBMZ induces CD4⁺ and CD8⁺ T effector memory cells (Tems). Groups of BALB/c mice were IP vaccinated with sterile PBS vehicle (n = 5), 10⁷ CFUs S19 (n = 5), or 10⁷ CFUs znBMZ (n = 5) on day 0. On day 42, spleens from individual mice were harvested and mononuclear cells were isolated for evaluation T cell subsets by flow cytometry. (A) Flow cytometry plots reveal that S19-vaccinated mice elicit mostly CD4⁺ T cells in contrast to both CD4⁺ and CD8⁺ T cells induced with subsequent znBMZ vaccination. (B) Total CD4⁺ and (C) CD8⁺ T cells are depicted. The frequencies of (D) CD4⁺ and (F) CD8⁺ T cells were evaluated for induction of Tems by staining splenic lymphocytes for CD44^High CD62^Low CX3CR1⁺ KLRG1⁺ T cells and (E) total CD4⁺ and (G) CD8⁺ Tems were quantified. Values are the mean total T cells and Tems from individual mice ± SEM; *** p < 0.001, differences vs. PBS-treated mice; ^††† p < 0.001 vs. S19-vaccinated mice.

Figure 5

IP vaccination with znBMZ induces IFN-γ-producing CD4⁺ and CD8⁺ T cells. The spleens from the same groups of mice evaluated in Figure 4 were further analyzed for IFN-γ⁺ T cells by flow cytometry. (A) Gating on CD4⁺ Tems, these were further analyzed for expression of IFN-γ. (B) The total number of IFN-γ⁺ CD4⁺ T cells and (C) IFN-γ⁺ Tems are depicted. (D) Gating on CD8⁺ Tems, these were further analyzed for expression of IFN-γ. (E) The total number of IFN-γ⁺ CD8⁺ T cells and (F) IFN-γ⁺ Tems are shown. Values are the mean total CD4⁺ and CD8⁺ T cells and CD4⁺ and CD8⁺ Tems from individual mice ± SEM; *** p < 0.001, ** p ≤ 0.002, * p < 0.05 differences vs. PBS-treated mice; ^††† p < 0.001, ^†† p ≤ 0.002 vs. S19-vaccinated mice.

Figure 6

A single IP vaccination with S19 or znBMZ elicits increased splenic IFN-γ and TNF-α, but no IL-17 following in vitro restimulation. Isolated splenic mononuclear cells from individual mice, described in Figure 4 and Figure 5, were restimulated in vitro with media or 10⁹ CFUs HKRB51 for three days. Harvested supernatants from duplicate cultures were analyzed for (A) IFN-γ, (B) TNF-α, and (C) IL-17. Values are the mean cytokines from duplicate cultures corrected for media only levels from individual mice ± SEM; *** p < 0.001, differences vs. PBS-treated mice; ^††† p < 0.001 vs. S19-vaccinated mice.