The Brucella abortus S19 DeltavjbR live vaccine candidate is safer than S19 and confers protection against wild-type challenge in BALB/c mice when delivered in a sustained-release vehicle

Abstract

Brucellosis is an important zoonotic disease of nearly worldwide distribution. Despite the availability of live vaccine strains for bovine (S19, RB51) and small ruminants (Rev-1), these vaccines have several drawbacks, including residual virulence for animals and humans. Safe and efficacious immunization systems are therefore needed to overcome these disadvantages. A vjbR knockout was generated in the S19 vaccine and investigated for its potential use as an improved vaccine candidate. Vaccination with a sustained-release vehicle to enhance vaccination efficacy was evaluated utilizing the live S19 DeltavjbR::Kan in encapsulated alginate microspheres containing a nonimmunogenic eggshell precursor protein of the parasite Fasciola hepatica (vitelline protein B). BALB/c mice were immunized intraperitoneally with either encapsulated or nonencapsulated S19 DeltavjbR::Kan at a dose of 1 x 10(5) CFU per animal to evaluate immunogenicity, safety, and protective efficacy. Humoral responses postvaccination indicate that the vaccine candidate was able to elicit an anti-Brucella-specific immunoglobulin G response even when the vaccine was administered in an encapsulated format. The safety was revealed by the absence of splenomegaly in mice that were inoculated with the mutant. Finally, a single dose with the encapsulated mutant conferred higher levels of protection compared to the nonencapsulated vaccine. These results suggest that S19 DeltavjbR::Kan is safer than S19, induces protection in mice, and should be considered as a vaccine candidate when administered in a sustained-release manner.

FIG. 1.

Survival of the B. abortus S19 ΔvjbR::Kan mutant in J774A.1 macrophages. Wild-type strain 2308, S19, and the B. abortus S19 ΔvjbR::Kan mutant were used to infect J774A.1 macrophages at an MOI of 1:100. After 30 min of incubation followed by 1 h of treatment with gentamicin, infected macrophages were further incubated for 0 or 48 h. Treated cells were lysed, serially diluted, and plated on TSA or TSA-kanamycin plates for CFU determination. The results are represented as the means of three independent experiments ± SEMs. Statistical differences were analyzed by ANOVA followed by Tukey's posttest comparing all groups to one another. *, P < 0.001 compared to the 2308 control; #, P < 0.05 compared to S19.

FIG. 2.

Clearance of B. abortus S19 ΔvjbR::Kan after infection. BALB/c mice (n = 5/time point) were infected with 1 ×10⁶ CFU/mouse of wild-type 2308 or S19 ΔvjbR::Kan. At 1, 3, 5, 7, and 9 weeks postinfection, mice were euthanized and the spleens were assessed for bacterial colonization (A) and weight (B). Values are the means of the results for individual mice ± standard errors of the means. Differences were determined by two-tailed Student's t test comparing S19 to the mutant (*, P < 0.05). The solid line in panel A represents the limit of detection, which is ≥5 CFU.

FIG. 3.

Spleen morphology in BALB/c mice vaccinated with S19 ΔvjbR::Kan. Mice were inoculated with 1 × 10⁵ CFU of either B. abortus 2308 (A), S19 ΔvjbR::Kan (B), S19 (C), or PBS (D). Animals were euthanized 3 weeks postinoculation, and spleens were weighed, harvested, and fixed for histological analysis.

FIG. 4.

Histological analysis of livers and spleens from BALB/c mice inoculated with S19, B. abortus 2308, S19 ΔvjbR::Kan, or PBS. In the livers, random distribution of inflammatory foci in mice vaccinated with either S19 (A) or 2308 (B) was observed (×10 magnification; ×40 magnification in the inset). Normal liver appearance was observed in mice inoculated with either S19 ΔvjbR::Kan (C) or PBS (D). Foci of inflammation are composed primarily of histiocytes. Scale bars in panels A and B, 100 μM; inset scale bars, 25 μM. In the spleens of mice vaccinated with either S19 (E) or 2308 (F), the white pulp is coalescing and enlarged, including marginal zones, and the formation of secondary lymphoid follicles is present (×4 magnification). There was minimal enlargement of the marginal zone in the mice vaccinated with either S19 ΔvjbR::Kan (G) or PBS (H). Scale bars in panels E to H, 200 μM.

FIG. 5.

Immunization efficacy and safety of B. abortus S19 ΔvjbR::Kan vaccine formulations. BALB/c mice were immunized i.p. with 1 × 10⁵ of either nonencapsulated or encapsulated S19 ΔvjbR::Kan. Control groups received empty capsules or S19. After 20 weeks, the mice were challenged i.p. with 1 × 10⁵ CFU wild-type 2308. At 1 week postchallenge, the mice were euthanized, their spleens harvested, and the bacterial loads (A) and spleen weights (B) determined. (A) Values are reported as the mean log₁₀ recovery of the 2308 challenge organism recovered from the spleens. Differences in colonization between all the groups were determined by ANOVA followed by a Tukey's posttest (*, P < 0.05; **, P < 0.001). (B) Spleen weights were measured in mg and were compared and analyzed by ANOVA followed by a Tukey's posttest comparing all groups to one another (*, P < 0.01; **, P < 0.001). For the statistical representation on both graphs, “a” symbolizes naïve animals, “b” S19-vaccinated animals, “c” encapsulated S19 ΔvjbR::Kan-vaccinated animals, and “d” nonencapsulated S19 ΔvjbR::Kan-vaccinated animals.

FIG. 6.

IgG1 and IgG2 anti-Brucella antibodies in serum from mice immunized with S19 ΔvjbR::Kan. BALB/c mice were inoculated i.p. with 1 × 10⁵ CFU of either nonencapsulated S19 ΔvjbR::Kan or encapsulated S19 ΔvjbR::Kan. Mice within the control group received empty capsules in lieu of vaccine. At 0, 3, 7, and 21 weeks postvaccination (1 week postchallenge), serum samples were collected for IgG1 (A) and IgG2 (B) determination by ELISA. Results are shown as the means ± SEM of absorbance at 450 nm. For the statistical representation on both graphs, “a” symbolizes naïve animals, “b” encapsulated S19 ΔvjbR::Kan-vaccinated animals, and “c” nonencapsulated S19 ΔvjbR::Kan-vaccinated animals.