DeltaznuADeltapurE Brucella abortus 2308 mutant as a live vaccine candidate

Abstract

To create a new, safe brucellosis live vaccine, a double mutant strain was constructed from Brucella abortus 2308. Using the DeltaznuA B. abortus 2308 mutant, a second mutation was introduced by deleting purE gene. The DeltaznuA DeltapurE B. abortus 2308 strain was less capable of surviving in macrophages. When evaluated in vivo, it was cleared within 8 weeks (wks) from mice, causing significantly less inflammation than spleens obtained from wild-type B. abortus 2308-infected mice. Furthermore, two doses of DeltaznuA DeltapurE B. abortus 2308 conferred 0.79 log protection, similar to S19 as did a single dose of DeltaznuA B. abortus 2308. Thus, this study shows the DeltaznuA DeltapurE B. abortus 2308 strain to be a potential livestock vaccine candidate.

Fig. 1.

Schematic illustration of purE knockout mutation from mutant strain, ΔznuA B. abortus 2308. The orientation of the purE and its surrounding genes are indicated by large solid arrows. The orientation of the different primers used in this study are indicated by small arrows. The position of in-frame deletion of a 471 bp purE inner DNA fragment from ΔznuA B. abortus 2308 during the construction of the purE mutant by allelic replacement is indicated.

Fig. 2.

The ΔznuAΔpurE B. abortus 2308 mutant is readily cleared from RAW264.7 macrophages. (A–C) Varying infection ratios of wild-type strain 2308, S19 vaccine, ΔznuA 2308 mutant, and the ΔznuAΔpurE 2308 mutant were used to infect RAW264.7 macrophages for 4, 24, or 48 h. Values are the means of two independent experiments ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 depict significant differences in strain 2308 colonization of macrophages versus S19, ΔznuA mutant, or ΔznuAΔpurE double mutant; ^#P < 0.05; ^##P ≤ 0.01; ^###P < 0.001 depict significant differences in S19 colonization versus ΔznuA or ΔznuAΔpurE mutants; ^§P < 0.05; ^§§P ≤ 0.01; ^§§§P < 0.001 depict significant differences in ΔznuA colonization versus ΔznuAΔpurE mutant.

Fig. 3.

ΔznuAΔpurE B. abortus 2308 is completely cleared by 8 wks post-infection. BALB/c mice were i.p. dosed with 1 × 10⁸ CFU of wild-type B. abortus 2308 or ΔznuAΔpurE mutant. At 2, 4, 8, and 12 wks interval, individual spleens were assessed for colonization. Values are the means of individual mice ± SD, and differences in splenic weight and colonization were determined via unpaired t test (*P < 0.05; **P = 0.01).

Fig. 4.

ΔznuAΔpurE B. abortus 2308 vaccine protects against wild-type B. abortus challenge. (A and B) BALB/c mice were dosed with 1 × 10⁹ (n = 8) or 1 × 10⁸ (n = 8) CFUs of either ΔznuAΔpurE 2308 or ΔznuA 2308 vaccines. Additional groups of mice were also dosed twice with 1 × 10⁸ CFUs of ΔznuAΔpurE 2308 (n = 13) or ΔznuA 2308 (n = 4) vaccines. As a positive vaccination control, one group of mice was vaccinated with 1 × 10⁸ S19 (n = 10); as a negative vaccination control, one group of mice was dosed i.p. with sterile PBS (sPBS; n = 9). After 8 wks, mice were challenged with 5 × 10⁴ CFU wild-type B. abortus 2308. Four wks post-challenge, their spleens were assessed for changes in (A) weight and (B) CFU levels. Values are the means of individual mice SEM from two independent experiments. *P < 0.001; **P ≤ 0.02; ***P < 0.05 depict significant differences between vaccinated and PBS-dosed mice; ^¶P < 0.001; ^¶¶P ≤ 0.02; ^¶¶¶P < 0.05 depict significant differences between ΔznuAΔpurE B. abortus 2308-vaccinated mice versus other vaccinated mice; and ^§P < 0.001 depicts significant differences in splenic weights versus S19-vaccinated mice.