Extended safety and efficacy studies of the attenuated Brucella vaccine candidates 16 M(Delta)vjbR and S19(Delta)vjbR in the immunocompromised IRF-1-/- mouse model

Abstract

The global distribution of brucellosis and high incidence in certain areas of the world warrant the development of a safer and efficacious vaccine. For the past 10 years, we have focused our attention on the development of a safer, but still highly protective, live attenuated vaccine for human and animal use. We have demonstrated the safety and protective efficacy of the vaccine candidates 16 MΔvjbR and S19ΔvjbR against homologous and heterologous challenge in multiple immunocompetent animal models, including mice and deer. In the present study, we conducted a series of experiments to determine the safety of the vaccine candidates in interferon regulatory factor-1-knockout (IRF-1(-/-)) mice. IRF-1(-/-) mice infected with either wild-type Brucella melitensis 16 M or the vaccine strain Brucella abortus S19 succumb to the disease within the first 3 weeks of infection, which is characterized by a marked granulomatous and neutrophilic inflammatory response that principally targets the spleen and liver. In contrast, IRF-1(-/-) mice inoculated with either the 16 MΔvjbR or S19ΔvjbR vaccine do not show any clinical or major pathological changes associated with vaccination. Additionally, when 16 MΔvjbR- or S19ΔvjbR-vaccinated mice are challenged with wild-type Brucella melitensis 16M, the degree of colonization in multiple organs, along with associated pathological changes, is significantly reduced. These findings not only demonstrate the safety and protective efficacy of the vjbR mutant in an immunocompromised mouse model but also suggest the participation of lesser-known mechanisms in protective immunity against brucellosis.

Fig 1

Temperature profiles from infected immune-incompetent mice. IRF-1^−/− mice were implanted with transponders and monitored daily over a 33-day period following inoculation with 1 × 10⁶ CFU/mouse of either 2308 (A), S19 (B), S19ΔvjbR (C), 16M (D), or 16MΔvjbR (E). The dotted lines denote the day of inoculation; temperature profiles to the left of the dotted lines denote basal body temperatures prior to inoculation. Each symbol represents an individual mouse tracked for 30 days postinoculation.

Fig 2

Health checks of immune-incompetent mice. Differences in behavior of IRF-1^−/− mice infected with 1 × 10⁶ CFU/mouse of either 16MΔvjbR (A) or 16M (B) were noticeable by day 10 postinoculation. Note the abnormal posture and ruffled coat in the mouse inoculated with the wild-type strain.

Fig 3

Survival of IRF-1^−/− mice infected with 1 × 10⁶ CFU/mouse of either 2308, S19, S19ΔvjbR, 16M, or 16MΔvjbR. Mice inoculated with ΔvjbR vaccine candidates survived longer than mice inoculated with either 16M (P < 0.005), 2308 (P < 0.005), or S19 (P < 0.005).

Fig 4

Bacterial colonization in the spleens and livers in terminally ill IRF-1^−/− mice inoculated with 1 × 10⁶ CFU/mouse of either 16M (A and B), 2308 (C and D), or S19 (E and F). The dotted lines denote 30 days postinfection, and animals that did not succumb to the infection prior to this time point were euthanized to determine the extent of bacterial colonization. Data are expressed as log₁₀ number of CFU/organ.

Fig 5

Spleen and liver weights in terminally ill IRF-1^−/− mice inoculated with 1 × 10⁶ CFU/mouse of either 2308 (A), S19 (B), or 16M (C). Each point represents an individual mouse and the spleen or liver weight at the time of death. The dotted lines represent the average normal weight of each organ for naïve animals.

Fig 6

Gross pathological changes observed at day 30 in mice infected with 1 × 10⁶ CFU/mouse of either 16M (A) or 16MΔvjbR (B). Note the markedly enlarged and pale liver and spleen in the mouse inoculated with 16M. The abdominal cavity in the mouse inoculated with the 16MΔvjbR was unremarkable.

Fig 7

Microscopic changes observed in the spleens (A to F) and livers (G to L) of IRF-1^−/− mice infected with either 16MΔvjbR (A and G), 16M (B and H), 2308 (C and I), S19 (D and J), or S19ΔvjbR (E and K) or naïve mice (F and L). Note the complete obliteration of the normal splenic parenchyma and replacement with a histiocytic infiltrate in 16M-, 2308-, and S19-inoculated mice (B, C, and D). None of the mice infected with the vjbR mutants exhibited significant changes in the spleen. All livers inoculated with 16M, 2308, or S19 (H, I, and J) exhibited multifocal, random neutrophilic and histiocytic infiltrates. The livers from ΔvjbR mutant-infected mice are unremarkable.

Fig 8

16M recovery from spleens and livers 1 week postchallenge in IRF-1^−/− mice. Groups of four mice were vaccinated with 1 × 10⁶ of either 16MΔvjbR or S19ΔvjbR for 8 weeks prior to challenge. Data are expressed as log₁₀ number of CFU/organ in the spleens (A) or livers (B).

Fig 9

Spleen (A) and liver (B) weights of IRF-1^−/− mice vaccinated with either 16MΔvjbR or S19ΔvjbR and challenged intraperitoneally with 16M. Data represent the average of 4 mice per group.

Fig 10

Temperature profiles collected over a 1-week period from IRF-1^−/− mice vaccinated with 1 × 10⁶ CFU/mouse of either S19ΔvjbR or 16MΔvjbR and challenged at 8 weeks postvaccination with 1 × 10⁶ CFU/mouse of wild-type B. melitensis 16M.

Fig 11

Microscopic changes observed in the spleens (A, C, E, and G) or livers (B, D, F, and H) of IRF-1^−/− mice vaccinated with either S19ΔvjbR (E and F) or 16MΔvjbR (G and H) and challenged at 8 weeks postvaccination with 1 × 10⁶ CFU/mouse of wild-type B. melitensis 16M. Results for naïve but challenged mice (C and D) and naïve mice (A and B) are presented for comparison. Note the marked reduction in the inflammatory response in both the spleen and liver in animals that received the ΔvjbR mutants.