Immunology and Cell Biology (2007) 85, 551–557 & 2007 Australasian Society for Immunology Inc. All rights reserved 0818-9641/07 $30.00 www.nature.com/icb
ORIGINAL ARTICLE
Development of protective immunity in a murine model of melioidosis is influenced by the source of Burkholderia pseudomallei antigens Jodie L Barnes and Natkunam Ketheesan Melioidosis is a potentially fatal disease caused by the bacterium, Burkholderia pseudomallei. The current study was carried out to determine the mechanisms involved in the development of protective immunity in a murine model of melioidosis. Following intravenous infection with B. pseudomallei, both C57BL/6 and BALB/c mice demonstrated delayed-type hypersensitivity responses and lymphocyte proliferation towards B. pseudomallei antigens, indicating the generation of B. pseudomalleispecific lymphocytes. Adoptive transfer of these lymphocytes to naı¨ve C57BL/6 mice was demonstrated by a delayed-type hypersensitivity response. Mice were not protected from a subsequent lethal challenge with a highly virulent strain of B. pseudomallei, suggesting that a single intravenous dose of the bacterium is insufficient to induce a protective adaptive immune response. Attempts to induce resistance in susceptible BALB/c mice used repetitive low-dose exposure to live B. pseudomallei. Immune responses and resistance following subcutaneous immunization with live B. pseudomallei were compared with exposure to heat-killed, culture filtrate and sonicated B. pseudomallei antigens. Compared to heat-killed B. pseudomallei, significant protection was generated in BALB/c mice following immunization with live bacteria. Our studies also demonstrate that the type of immune response generated in vivo is influenced by the antigenic preparation of B. pseudomallei used for immunization. Immunology and Cell Biology (2007) 85, 551–557; doi:10.1038/sj.icb.7100084; published online 12 June 2007 Keywords: BALB/c; Burkholderia pseudomallei; C57BL/6; cell-mediated immunity; melioidosis; protective immunity
The emerging tropical infectious disease, melioidosis, is caused by the soil bacterium Burkholderia pseudomallei. Melioidosis is considered endemic in regions of Southeast Asia and northern Australia, with incidence rates increasing following periods of heavy rainfall. A protective vaccine against B. pseudomallei infection is not yet available. Clinical manifestations of melioidosis range from a rapidly fatal septicemic illness to a subclinical state, identified only by seroconversion in an infected individual.1 B. pseudomallei is a facultative intracellular bacterium that can cause latent infection through its persistence in phagocytic and nonphagocytic cells of the host.2 Reactivation of latent melioidosis to clinical disease is well documented following periods of immunosuppression.3 Therefore, cellmediated immune (CMI) responses are considered to play an important role in controlling infection with this bacterium. An adaptive CMI response has been demonstrated in culture-confirmed melioidosis patients4 and two studies recently described high CMI responses in individuals with subclinical melioidosis.5,6 In experimental melioidosis, several studies have demonstrated an increase in TH1-type cytokines during the early stages of infection.7–10 Resistance to infection with B. pseudomallei is governed by the production of interferon (IFN)-g from various cellular sources within the first
24 h.7,9 A CMI response, primarily involving CD4+ T cells, was also recently demonstrated in BALB/c mice that had been infected intraperitoneally (i.p.) with B. pseudomallei.11 We12–14 and others8,15 have begun to investigate potential immunological mechanisms underlying the differential susceptibility observed in the BALB/c-C57BL/6 model of human melioidosis. Compared to C57BL/6 mice, B. pseudomallei infection of BALB/c mice results in hyperproduction of proinflammatory cytokines,10 overwhelming septicemia, wide-spread tissue necrosis and subsequent death.13 Disparate expression of messenger RNA for several chemokines and colony-stimulating factors between BALB/c and C57BL/6 mice is also associated with differences in bacterial growth and the composition of cellular infiltrate.12 While these previous studies support a role for CMI responses, most pathways for the development of protective immunity of the host toward B. pseudomallei have not yet been identified. The nature of a protective host immune response, and the conditions under which it is induced, are fundamental for improved clinical management of patients and vaccine development. Therefore, using our previously characterized mouse model of melioidosis,13 the major focus of the current study was to demonstrate the involvement of CMI responses in the development of protective immunity in melioidosis.
Department of Microbiology and Immunology, School of Veterinary and Biomedical Sciences, James Cook University, Townsville, Queensland, Australia Correspondence: Dr JL Barnes, Department of Microbiology and Immunology, School of Veterinary and Biomedical Sciences, James Cook University, Townsville, Queensland, Australia 4811. E-mail:
[email protected] Received 12 April 2007; revised 9 May 2007; accepted 15 May 2007; published online 12 June 2007
Development of protection in melioidosis JL Barnes and N Ketheesan 552
After establishing the presence of B. pseudomallei-specific mononuclear leukocytes (MNL) in BALB/c and C57BL/6 mice exposed to the bacterium, we investigated the ability of these cells to provide resistance in naı¨ve mice using adoptive transfer assays. Adoptive transfer experiments were performed using C57BL/6 mice due to their relative resistance to B. pseudomallei infection. Since the results of these studies demonstrated that exposure to a single small dose of B. pseudomallei via the intravenous (i.v.) route is insufficient to generate a protective host immune response, alternative methods of immunization were subsequently employed. For a variety of antigens, the administrated dose is crucial for determining the type of immunity that is induced16,17 and studies of adaptive immunity to several intracellular bacteria, such as Mycobacterium tuberculosis and Listeria monocytogenes, have demonstrated that a protective immune response is generated only in the presence of the living organism, since it is predominantly mediated by secreted antigens.18,19 Therefore, we investigated whether resistance to B. pseudomallei could be increased in susceptible BALB/c mice by repetitive subcutaneous (s.c.) injection of low doses of bacteria. We also compared protection afforded by immunization with live and killed preparations of B. pseudomallei. RESULTS Uptake and killing of B. pseudomallei, NCTC 13179 is more efficient in macrophages from C57BL/6 mice No differences in nitrite (NO2) levels were observed between peritoneal exudates cells (PEC) and IFN-g-primed PEC from C57BL/6 and BALB/c mice (data not shown). In contrast, uptake (17.5±2.6 versus 1.9±0.1%) and killing (80.8±3.3 versus 72.5±1.5%) of intracellular NCTC 13179 was significantly higher in PEC cultures from C57BL/6 mice compared to BALB/c mice. CMI responses develop in both BALB/c and C57BL/6 mice following intravenous immunization with B. pseudomallei Compared to controls, significantly greater footpad swelling and lymphocyte proliferation were observed in BALB/c (Po0.05) and C57BL/6 (Po0.05) mice previously exposed to B. pseudomallei (Table 1). Levels of IFN-g and interleukin (IL)-4 were measured in supernatants of MNL cultures derived from control and i.v.-immunized BALB/c and C57BL/6 mice, following stimulation with lipopolysaccharide (LPS)-neutralized B. pseudomallei lysate (BpLy1) (Table 1). Low numbers of IFN-g-producing MNL were detected in cultures stimulated with BpLy1 (Table 1). No differences were observed between IFN-g production in cultures from control mice compared to BALB/c (P40.05) or C57BL/6 (P40.05) mice immunized with B. pseudomallei. Similarly, there were no significant
differences between the two mouse strains in the numbers of IFN-gproducing cells (P40.05). For C57BL/6 mice, IL-4-producing MNL were more numerous in BpLy1 stimulated cultures derived from immunized mice compared to controls (Po0.05; Table 1). In contrast, no differences were observed in IL-4 production in MNL cultures from control or immunized BALB/c mice (Table 1). B. pseudomalleiexposed C57BL/6 mice had significantly higher numbers of IL-4producing lymphocytes compared to B. pseudomallei-exposed BALB/c mice (Po0.05). Adoptive transfer of MNL from C57BL/6 mice exposed to B. pseudomallei via the intravenous route does not provide protection against a lethal challenge A significant (Po0.05) delayed-type hypersensitivity (DTH) response was observed following adoptive transfer of splenic MNL from B. pseudomallei-exposed mice to naı¨ve mice compared to the adoptive transfer of MNL from control groups of mice (Po0.05; Figure 1a). Adoptive transfer of splenic MNL from B. pseudomallei-immunized mice did not provide protection against subsequent challenge with NCTC 13178, as indicated by the comparable survival rates of these mice (60%) with mice that received phosphate-buffered saline (PBS) only (60%; data not shown) at day 10 post-challenge. Similarly, no differences were observed in the bacterial loads (Figure 1b) of mice that were adoptively transferred with MNL from B. pseudomalleiexposed mice, PBS-control mice or PBS-only (no MNL) mice. Subcutaneous low-dose immunization of susceptible BALB/c mice with B. pseudomallei induces protection against subsequent lethal challenge There was a significant DTH response to BpLy1 at 48 h in BALB/c mice immunized with live B. pseudomallei, compared to PBS-control mice (Figure 2a; Po0.05). Although increased swelling was also observed in BpLy1-immunized mice, this response was not significantly greater than controls. Similarly, compared to controls, significant lymphocyte proliferation in response to BpLy1 was observed in MNL cultures derived from B. pseudomallei- and BpLy1-immunized BALB/c mice (Figure 2b; Po0.05 and Po0.05, respectively). However, the difference in proliferation of lymphocytes from B. pseudomalleiimmunized versus BpLy1-immunized mice was not significant. IgG2a and IgG1 (Figure 3a; Po0.05) levels were significantly increased in BpLy1-immunized mice compared to other treatment groups. An increase in IgG2a levels was also observed for B. pseudomallei-immunized mice; however, these levels were not significantly greater than levels in control mice. Serum levels of IgG2a and IgG1 were measured at day 10 in control and immunized BALB/c mice
Table 1 Delayed-type hypersensitivity, lymphocyte proliferation and cytokine responses in BALB/c and C57BL/6 mice in response to BpLy1 antigens Parameter
Test group
BALB/c
C57BL/6
DTH (change in footpad thickness; mm±s.e.m.)
Control
0.17±0.1
0.15±0.1
Lymphocyte proliferation (SI±s.e.m.)
Immunized Control
0.4±0.1* 3.67±0.8
0.5±0.1* 1.31±0.2
IFN-g (mean number of spots±s.e.m.)
Immunized Control
45.06±7.6* 3.89±3.9
27.85±1.9* 12.34±6.7
IL-4 (mean number of spots±s.e.m.)
Immunized Control
0±0 6.33±3.2
7.45±3.8 20.0±3.2
Immunized
18.67±5.9
Abbreviations: DTH, delayed-type hypersensitivity; IFN, interferon; IL; interleukin. *Indicated a significant difference in immunized mice (Po0.05) compared to controls.
Immunology and Cell Biology
89.33±12.0*
Development of protection in melioidosis JL Barnes and N Ketheesan
0.8
Control
*P
