Deletion of Two Genes in Burkholderia pseudomallei MSHR668 That Target Essential Amino Acids Protect Acutely Infected BALB/c Mice and Promote Long Term Survival

Abstract

Melioidosis is an emerging disease that is caused by the facultative intracellular pathogen Burkholderia pseudomallei. It is intrinsically resistant to many antibiotics and host risk factors play a major role in susceptibility to infection. Currently, there is no human or animal vaccine against melioidosis. In this study, multiple B. pseudomallei MSHR668 deletion mutants were evaluated as live attenuated vaccines in the sensitive BALB/c mouse model of melioidosis. The most efficacious vaccines after an intraperitoneal challenge with 50-fold over the 50% median lethal dose (MLD₅₀) with B. pseudomallei K96243 were 668 ΔhisF and 668 ΔilvI. Both vaccines completely protected mice in the acute phase of infection and showed significant protection (50% survivors) during the chronic phase of infection. The spleens of the survivors that were examined were sterile. Splenocytes from mice vaccinated with 668 ΔhisF and 668 ΔilvI expressed higher amounts of IFN-γ after stimulation with B. pseudomallei antigens than splenocytes from mice vaccinated with less protective candidates. Finally, we demonstrate that 668 ΔhisF is nonlethal in immunocompromised NOD/SCID mice. Our results show that 668 ΔhisF and 668 ΔilvI provide protective cell-mediated immune responses in the acute phase of infection and promote long term survival in the sensitive BALB/c mouse model of melioidosis.

Keywords: auxotroph; live attenuated vaccine; melioidosis.

Figure 1

BALB/c mice vaccinated with 668 ΔhisF and 668 ΔilvI are protected similarly during the acute phase of infection with B. pseudomallei K96243. Ten mice in each group were vaccinated twice (s.c.) with 1 × 10⁶ CFU in 0.1 mL of the respective mutant 21 days apart. Thirty days after the boost vaccination, the mice were challenged i.p. with 3.1 × 10⁶ CFU (50 MLD₅₀s) of B. pseudomallei K96243, and challenged mice were followed for 60 days. Each group of 10 mice was tested twice in most cases, and the Figure is a composite of the survival of mice vaccinated twice with the respective whole cell vaccine before challenge. p-values comparing the mean survival time between the PBS control and other vaccines: * p = 0.0006; ** p = 0.0145 (576 ΔilvI).

Figure 2

Inactivated B. pseudomallei K96243 (BpK) whole-cell vaccines do not protect mice as well as a MSHR668-derived auxotroph. Ten mice in each group were vaccinated with 50 µg of formalin-inactivated (fBpK), irradiated-inactivated (IRBpK), or a mixture of fBpK (50 µg) and IRBpK (50 µg) twice (s.c.) three weeks apart, and mice were challenged four weeks later (i.p.) with 2.5 × 10⁶ CFU (40 MLD₅₀s) of K96243. At the same time, PBS and live attenuated 668 ΔhisF ΔA0269 (1 × 10⁶ CFU) control groups were vaccinated twice (n = 10 in each group) and challenged at the same time. All mice were observed for 60 days after challenge. p-values comparing the mean survival time between the PBS control and other vaccines.

Figure 3

Cell-mediated immune responses were induced by both live and inactivated whole-cell vaccines, but live vaccines gave better protection in BALB/c mice. In most cases each vaccine was tested twice, and we show a representative of one study. N was 3–4 for each vaccine shown. (A) Splenocytes from mice vaccinated with live auxotrophs (1 × 10⁶ CFU) of 668 ΔilvI and 668 ΔhisF and derivatives (as described in Figure 1) expressed interferon (IFN)-γ upon restimulation with IRBpK. (B) Splenocytes from mice vaccinated with 50 µg of formalin-inactivated BpK (fBpK), irradiation-inactivated (IRBpK, or a mixture of fBpK and IRBpK twice, 21 days apart (as described in Figure 2), and spleens were prepared from vaccinated mice four weeks after the second vaccination. Significant differences in the amount of IFN-γ expressed between the stimulated splenocytes from PBS vaccinated mice and stimulated splenocytes from test vaccines are shown in A and B. In B we also show a comparison between the stimulated 668 ΔhisF ΔA0269 splenocytes verses other stimulated vaccines including the stimulated PBS control splenocytes.

Figure 3

Cell-mediated immune responses were induced by both live and inactivated whole-cell vaccines, but live vaccines gave better protection in BALB/c mice. In most cases each vaccine was tested twice, and we show a representative of one study. N was 3–4 for each vaccine shown. (A) Splenocytes from mice vaccinated with live auxotrophs (1 × 10⁶ CFU) of 668 ΔilvI and 668 ΔhisF and derivatives (as described in Figure 1) expressed interferon (IFN)-γ upon restimulation with IRBpK. (B) Splenocytes from mice vaccinated with 50 µg of formalin-inactivated BpK (fBpK), irradiation-inactivated (IRBpK, or a mixture of fBpK and IRBpK twice, 21 days apart (as described in Figure 2), and spleens were prepared from vaccinated mice four weeks after the second vaccination. Significant differences in the amount of IFN-γ expressed between the stimulated splenocytes from PBS vaccinated mice and stimulated splenocytes from test vaccines are shown in A and B. In B we also show a comparison between the stimulated 668 ΔhisF ΔA0269 splenocytes verses other stimulated vaccines including the stimulated PBS control splenocytes.

Figure 4

The 668 ΔhisF auxotroph was nonlethal in NOD/SCID mice. NOD/SCID mice (n = 10 for each group) that have impaired T- and B-cells and defective natural killer (NK) cells were inoculated (i.p.) with 668 ΔhisF (10,270 CFU), Bp82 (ΔpurM) control group (11,330 CFU), and MSHR668 wild-type (12,870 CFU). After inoculation, mice were observed for 21 days. Comparison of mean survival time between MSHR668 and 668 ΔhisF or Bp82 was p = 0.0002.