Vaccine 32 (2014) 5983–5988

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Comparison of the protective effects of killed Burkholderia pseudomallei and CpG oligodeoxynucleotide against live challenge Apichaya Puangpetch a,e , Robert Anderson b,c , Yan Y. Huang b,c , Rojana Saengsot a,e , Rasana W. Sermswan d,e , Surasakdi Wongratanacheewin a,e,∗ a

Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand Department of Microbiology & Immunology, Canadian Center for Vaccinology, Dalhousie University, Halifax, Nova Scotia, Canada c Department of Pediatrics, Canadian Center for Vaccinology, Dalhousie University, Halifax, Nova Scotia, Canada d Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand e Melioidosis Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand b

a r t i c l e

i n f o

Article history: Received 27 May 2014 Received in revised form 22 July 2014 Accepted 15 August 2014 Available online 16 September 2014 Keywords: CpG oligodeoxynucleotide Burkholderia pseudomallei Paraformaldehyde-killed Burkholderia pseudomallei Heat-killed Burkholderia pseudomallei

a b s t r a c t Melioidosis is a fatal disease caused by Burkholderia pseudomallei. Currently there is no vaccine available. Synthetic oligodeoxynucleotides with unmethylated CpG dinucleotide motifs (CpG ODN) can stimulate vertebrate immune cells and clear certain pathogens that are susceptible to a strong Th1 response. In our previous study, pretreatment with CpG ODN alone or CpG-ODN with cationic liposomes for 2–10 or 30 days before B. pseudomallei infection in mice conferred 80–100% protection. In the present study we investigated the protective effect of CpG-ODN together with heat-killed (HK) or paraformaldehyde-killed B. pseudomallei (PP). HK or PP were used to immunize BALB/c mice twice at 15-day intervals before intraperitoneal challenge with 5LD50 of B. pseudomallei and observed for 30 days. We found that PP could significantly protect mice (60%) with an increased survival time (24.8 ± 11.63 days) while in the HK and PBS groups, all infected mice died within 6 days. Although either CpG ODN or PP conferred significant protection, giving them in combination did not enhance it further. Serum IFN-␥ levels on day-5 (before challenge) of the PP and PP + CpG ODN groups were significantly higher than those of the PBS control group. The results further support the importance of IFN-␥ in host protection against B. pseudomallei and suggest further study on paraformaldehyde-killed bacteria as a component of a future B. pseudomallei vaccine. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Melioidosis is an infectious disease caused by Burkholderia pseudomallei. It is a major public health problem in Southeast Asia and tropical Australia. It shows a broad spectrum clinical profile, ranging from asymptomatic to fatal septicemia or chronic abscess formation [1,2]. In northeast Thailand, the disease shows a 40% mortality rate due to septicemia even after treatment. The incidence in 2006 was estimated to be 8.63 per 100,000 people [3,4] and 10% of relapse cases were found even with prolonged antibiotic treatments [5]. Currently, there is no vaccine available. B. pseudomallei infected C57BL/6 mice were more resistant to infection and showed lower bacteremia than BALB/c mice. This is

∗ Corresponding author at: Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen, 40002. Thailand. Tel.: +66 43 363808; fax: +66 43 348385. E-mail address: sura [email protected] (S. Wongratanacheewin). http://dx.doi.org/10.1016/j.vaccine.2014.08.035 0264-410X/© 2014 Elsevier Ltd. All rights reserved.

because of the stronger Th1 response in C57BL/6 mice [6]. IFN␥, IL-12 and TNF-␣, contributing to a Th1 cell-mediated immune response, were found to be early important mediators of protection since the administration of neutralizing antibodies against these cytokines increased susceptibility to infection [7]. However, the relative importance of the innate and the adaptive immune responses remain unclear in B. pseudomallei infection [8]. Development of B. pseudomallei vaccines using various formulations and routes of immunization and challenge has been reported [9]. The lipopolysaccharide (LPS), capsule polysaccharide, flagella [10–12] as well as several purified bacterial proteins, including the lipoprotein releasing system transmembrane protein (LolC), an adenosine triphosphate-binding cassette transporter protein [13], the type III secretion system protein [14] and two outer membrane proteins, Omp3 and Omp7 [15], have been used for immunization to prevent melioidosis. Live attenuated B. pseudomallei has also been shown to confer some protection [12,16,17]. In passive protection, monoclonal antibodies against capsular polysaccharide and lipopolysaccharide provided only partial protection as some

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bacteria were still found in various mouse organs after live challenge [18]. Bacterial outer membrane proteins present an interface for interaction with host cells that may play a role in bacterial adhesion and invasion into host cells. Bacterial surface proteins thus have potential as vaccine candidates [19,20]. Paraformaldehydekilled B. pseudomallei (PP) was shown to stimulate higher IL-12 release from human dendritic cells than heat-killed bacteria (Stitaya Sirisinha, 2006, unpublished data). Given appropriate adjuvants, such antigens may stimulate host protective immune responses. The adjuvant CpG ODN is a synthetic oligodeoxynucleotide that contains unmethylated cytosine–phosphate–guanosine, thereby mimicking invertebrate DNA which can stimulate the mammalian immune response. CpG ODN can promote a Th1 immune response by interacting with TLR9 during endocytosis, resulting in improved antigen uptake and presentation by APCs and subsequent elicitation of antibodies and T cell responses, as well as chemokine and cytokine secretion by B and T cells, NK cells, dendritic cells and monocytes [21,22]. Several reports indicate that CpG ODN can protect mice from certain bacterial, viral and parasitic infections [23–25]. Our group demonstrated that CpG ODN or CpG ODN with cationic liposomes given 2–10 or 30 days respectively before challenge could induce significant protection in mice against B. pseudomallei [26]. Since CpG ODN stimulates only innate immunity, we investigated here the inclusion of bacterial antigen to stimulate the adaptive immune response. These combinations were then evaluated for protective efficacy in BALB/c mice. 2. Materials and methods 2.1. Animals Male BALB/c mice aged 4–6 weeks were obtained from the National Animal Centre, Mahidol University. All mice were maintained in the animal unit at the Faculty of Medicine, Khon Kaen University and fed with water and standard laboratory diet. These BALB/c mice were used for B. pseudomallei infection under the approved protocol (No. AEKKU13/2550) from the animal ethics committee of the Faculty of Medicine, Khon Kaen University, Thailand. 2.2. CpG oligodeoxynucleotide (CpG ODN) The CpG ODN used in this study was CpG ODN 1826 (5 TCCATGACGTTCCTGACGTT3 ) which is classified as a B class ODN and possesses a nuclease-resistant phosphorothioate backbone. It was provided by Coley Pharmaceutical Group (Wellesley, MA, USA) and contained only a trace of endotoxin (less than 7.5 × 10−8 endotoxin units/ng), as determined by the Limulus assay (Whittaker Bioproducts, Walkersville, MD). 2.3. Bacterial strains and culture conditions Virulent B. pseudomallei strain A2 was isolated from a patient with sepsis melioidosis. This strain had an LD50 of 20 cells. The bacteria were streaked on Ashdown media agar and incubated at 37 ◦ C. A single colony was picked and inoculated into 3 ml Tryptic soy broth (TSB), and incubated at 37 ◦ C overnight. A one percent inoculum was transferred into 50 ml TSB and allowed to grow for 3 h with shaking at 250 rpm in a 37 ◦ C incubator. The bacterial cells were diluted in phosphate-buffered saline (PBS, pH 7.2) to a desired concentration. The exact number of viable bacteria in the suspension was determined by plating the bacteria on Ashdown media and counted after 30–48 h of incubation at 37 ◦ C and expressed as

CFU/ml. All procedures described were carried out in a biosafety biohazard hood. 2.4. Acute, convalescent and normal human serum samples Acute sera were collected from six adult patients presenting with acute culture-confirmed melioidosis, in the Srinagarind Hospital, Khon Kaen, Thailand. Convalescent sera were taken from the same patients (after antibiotic treatment for 12 weeks) following recovery from the disease. Normal human serum samples were collected from six healthy individuals in Khon Kaen province. Each group of serum was pooled. The protocol for collection of patient sera was approved by the Human Ethics Committee, Khon Kaen University, Khon Kaen, Thailand. 2.5. Paraformaldehyde-killed B. pseudomallei (PP) and heat-killed B. pseudomallei (HK) Aliquots of B. pseudomallei A2 (108 CFU/mouse) were suspended in PBS and treated with 10% paraformaldehyde for 10 min (PP) or heated at 100 ◦ C for 10 min (HK). Bacteria were washed three times with PBS and loss of cell viability was verified by spreading on Ashdown agar. The bacterial suspensions were stored at −20 ◦ C until used. 2.6. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting Heat-killed bacteria (HK) and paraformaldehyde-killed B. pseudomallei (PP) samples were boiled with sample buffer. Three microgram of HK and PP samples were loaded onto a 10% polyacrylamide gel, and after electrophoresis, the proteins were visualized by silver staining [27]. For Western blot analysis, proteins were transferred onto a nitrocellulose membrane using a Transblot semidry transfer cell (Bio-Rad Laboratories, CA) at 120 mA for 2 h. After blocking with 5% skim milk in Tris-buffered saline Tween20 (TBST) (20 mM Tris–base, 136 mM NaCl, 0.1% Tween 20) for 1 h at 37 ◦ C, membranes were washed three times with TBST before incubation with primary Ab (1:50 dilution of pooled acute and pooled convalescent melioidosis patient sera) followed by washing and treatment with secondary antibody (1:5000 dilution of goat anti-human IgG conjugated with horseradish peroxidase (Sigma, USA). Antibody-reactive proteins were visualized using the SuperSignal® West Pico Chemiluminescent substrate (PIERCE Biotechnology Inc., Rockford, IL, USA) followed by exposure to x-ray film. 2.7. Immunizations and challenge Groups of 10 (five per group in two independent experiments) mice were immunized via the intramuscular (i.m.) route with 108 cells of PP or 108 cells of HK twice over a 15 days interval (day −30 and day −15). After the last immunization, animals were challenged with B. pseudomallei A2 (5LD50 ) via the intraperitoneal (i.p.) route on day 0 and mice were monitored daily for mortality for 30 days. For the study of CpG ODN combined with PP, groups of 10 mice were immunized with the combination at a ratio of 100 ␮g CpG ODN and 108 cells of PP or PP alone via the i.m. route twice with a 15-day interval (day −30 and day −15). For positive control, 100 ␮g CpG ODN was injected 2 days before infection (day −2) and vaccine solution agent (PBS) was used for injection in the negative control group. After the last immunization, animals were challenged with B. pseudomallei (5LD50 ) via the i.p. route on day 0 and mice were monitored daily for mortality for 30 days. Mice showing severe distress were euthanized for humane reasons when possible. Blood was

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Fig. 1. Silver stained SDS-PAGE protein and immuno-reactive profiles of heat-killed and paraformaldehyde-killed B. pseudomallei. Silver stained SDS-PAGE of protein profiles (A). Lane M: protein standard marker (kDa), Lane 1: Heat-killed B. pseudomallei (HK) and lane 2: Paraformaldehyde-killed B. pseudomallei (PP). Three microgram protein from HK or PP sample supernatants were resolved on a 10% polyacrylamide gel and silver-stained. Western blot of the antigens recognized by pooled acute, convalescent human sera and normal human serum (B). Three microgram of HK or PP were resolved on a 10% polyacrylamide gel. Proteins were transferred onto a nitrocellulose membrane and probed with a 1:50 dilution of pooled acute and convalescent sera from six patients or normal human sera, followed by horseradish peroxidase-conjugated goat anti-human IgG (Sigma, USA) (1:5000 dilution). For detection of proteins, the SuperSignal® West Pico Chemiluminescent substrate (PIERCE Biotechnology Inc., Rockford, IL, USA) was used, followed by exposure to X-ray film. HK, heat-killed B. pseudomallei; PP, paraformaldehyde-killed B. pseudomallei. The molecular weights of immuno-reactive proteins are indicated.

collected from each animal via retroorbital puncture with sterile heparinized capillary tubes for cytokine and bacteremia determinations. The individual blood samples were cultured on Ashdown media and numbers of colonies, expressed as CFU/ml, were counted after 30–48 h of incubation at 37 о C. The colonies with appearance typical of B. pseudomallei were immunologically identified using the latex agglutination test kit specific for B. pseudomallei antigens [28]. 2.8. Measurement of specific IgG antibodies against B. pseudomallei by ELISA Specific IgG antibodies to B. pseudomallei were determined by ELISA. Microtiter plates (96-well; Nunc) were coated with 100 ␮l of 5 ␮g/ml of B. pseudomallei heat killed antigen, and incubated overnight at 4 ◦ C. Plates were washed and blocked with 2% skim milk in incubation buffer (150 mMNaCl, 9 mM Na2 HPO4 , 1.25 mM NaH2 PO4 ·2H2 O 0.15 g, 0.05% Tween 20). Serum samples were diluted to 1:200 with 1% skim milk in incubation buffer and applied to the plates. Secondary antibody was 1:2000 diluted HRP-conjugated anti-mouse IgG (AbD serotec, USA). The plate was washed five times in PBS-Tween; then 100 ␮l of freshly mixed tetramethylbenzidine (TMB) substrate solution was added and the plate incubated in the dark for 15 min with shaking. The reaction was stopped by adding with 100 ␮l 2 N H2 SO4 prior to measuring the OD450 nm within 30 min. 2.9. Measurement of mouse cytokines (IFN-) concentration by ELISA Mouse IFN-␥ ELISA assays were performed using commercial mouse IFN-␥ MAXTM Deluxe ELISA Kits (Biolegend, U.S.A) according to the manufacturer’s instructions. The detection limit of mouse IFN-␥ was 15.6 pg/ml. 2.10. Statistical analysis For parametric statistics, the independent T test was used for comparisons between two groups. Comparisons of more than two

groups were analyzed by one-way analysis of variance (ANOVA), followed by post hoc intergroup comparison. For nonparametric statistics, the Mann Whitney U test and Kruskal–Wallis analysis were used. Survival times were compared using Kaplan–Meier curves and the log-rank test from GraphPad Prism version 4.0, GraphPad Software (San Diego, CA). Data were considered statistically significant if the p value was less than 0.05. 3. Results 3.1. Antigenic characterization of paraformaldehyde-killed and heat-killed B. pseudomallei Antigenic characterization of PP and HK was performed using SDS-PAGE (Fig. 1A). Total proteins visualized after SDS-PAGE by silver staining demonstrated that HK (Fig. 1A, lane 1) showed a more complex protein pattern than PP (Fig. 1A, lane 2). The Western blot patterns probed with acute sera showed several bands from HK and only two bands in PP (Fig. 1B). Conversely, convalescent sera recognized three similar protein bands with molecular weights of 66.5, 30 and 20 kDa in PP and 66.5, 32 and 20 kDa in HK. Normal human sera reacted with 20 and 28 kDa proteins in HK and 57, 50 and 21 kDa proteins in PP. The 30 kDa protein in PP that reacted with convalescent but not in acute and normal sera may possibly represent a protein which induces protective antibodies. 3.2. Paraformaldehyde-killed B. pseudomallei confers better protection in mice against B. pseudomallei challenge than heat-killed B. pseudomallei We determined the protective response and bacterial load of PP or HK immunization in mice challenged with B. pseudomallei. Ten mice were included in each of the three experimental groups. In each group, mice were immunized by the i.m. route with PP and HK twice on days −30 and −15. Mice injected with PBS were used as a control. Mice were challenged on day 0 with 100 CFU of B. pseudomallei A2 in a 100 ␮l volume. As shown in Fig. 2A, 60 percent of the

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Fig. 2. Survival, bacteremia and B. pseudomallei-specific IgG of HK- or PP-immunized BALB/c mice after challenge with B. pseudomallei A2. Mice were immunized i.m. twice with a 15-day interval with 100 ␮l of HK, PP (108 cells/ml) or PBS as a negative control. Two weeks after the second immunization, mice were challenged i.p. with 100 CFU of B. pseudomallei A2. All mice were observed until 30 days after infection. Survival curves were plotted against time (A). The blood samples from each group were taken on day 2 after challenge. The bacteremia (B) and specific IgG antibodies against B. pseudomallei (C) were determined. HK, heat-killed B. pseudomallei; PP, paraformaldehyde killed B. pseudomallei and PBS, phosphate buffer saline. The asterisk (* ) indicates significant difference from control (p < 0.05). Data are from two independent experiments.

PP immunized mice survived until day 30, when the experiments were terminated and data were analyzed. The mean survival period for PP-immunized mice was significantly higher than those in the HK or PBS groups (Table 1). No significant difference was found in the percent of survival between the HK immunized and PBS injected groups (Table 1). Both the HK and the PBS control-immunized mice died within the first 6 days and the bacteremia in the PP group was significantly lower than that in the HK and PBS groups (Fig. 2B). We have shown previously that the protective effect of CpG on day −2 could protect the mice at least 3 months [26]. In our previous report, it was observed that various numbers of bacteria were still present in the spleen, liver and lungs of untreated, but not the CpGtreated, animals that were sacrificed on day 4, when the bacteria had already disappeared from the circulation [26]. The specific IgG to B. pseudomallei in PP and HK was significantly higher than PBS control (Fig. 2C). The PP therefore may be useful as a protective antigen against B. pseudomallei infection.

3.3. CpG ODN combined with paraformaldehyde-killed B. pseudomallei does not enhance the protection against B. pseudomallei infection In order to increase protection, CpG ODN was used as adjuvant together with PP. Mice were immunized twice with a 15 day interval with PP alone, CpG ODN + PP or CpG ODN alone on day −2 as a positive control, prior to challenge with B. pseudomallei A2. As shown in Fig. 3A, CpG ODN and PP-treated mice yielded 80% and 50% protection respectively (Table 1) whereas all PBS-treated mice died. In contrast, the combination of PP + CpG ODN did not offer any further protection (30% survival) (Fig. 3A). The mean survival times for the mice treated with CpG ODN, PP alone or PP + CpG ODN were significantly longer than mice treated with PBS (Table 1). The serum levels of IFN-␥ on day-5 (before challenge) after PP treatment showed high IFN-␥ production, which reached statistical significance, compared to the PBS group (Fig. 3B). This high serum

Table 1 Survival times and percent protection of mice immunized with PP, PP + CpG ODN, HK or PBS control. Experiment

Immunization protocol

Survival time (mean ± SD)

% Protection

Statistical significancea

I

PP on days −30, −15 HK on days −30, −15 PBS on days −30, −15 PP on days −30, −15 PP + CpG ODN days −30, −15 CpG ODN on day −2 PBS on days −30, −15

24.8 ± 11.63 5.4 ± 1.94 4.6 ± 1.34 23.7 ± 2.41 21 ± 2.44 25 ± 3.34 8.6 ± 0.79

60 0 0 50 30 80 0

p = 0.021b p = 0.651 NA p = 0.031b p = 0.042b p = 0.018b NA

II

All mice were challenged with 5LD50 of B. pseudomallei on Day 0. Ten mice per group. PP = paraformaldehyde-killed B. pseudomallei; HK = heat-killed B. pseudomallei; PBS = phosphate buffered saline; NA = not applicable. a Statistical tests were done compared to PBS control. b Indicates significant difference.

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Fig. 3. Protection and serum IFN-␥ levels of BALB/c mice immunized with PP or PP + CpG ODN and challenged with B. pseudomallei. Mice were immunized i.m. with 108 CFU of PP or 108 CFU of PP combined with CpG ODN on day −30 and day −15. CpG ODN or PBS administered on day −2 were used as positive and negative controls, respectively. All animals were then challenged i.p. with 5LD50 (100 CFU) B. pseudomallei A2 on day 0. Mice were observed daily and percentage survival was plotted against time (A). Blood was collected on day −23, −16, −5, 2, 7 for IFN-␥ determination (B). The results represent mean ± SE. Statistical analysis was performed using Kruskal–Wallis analysis following Mann Whitney U test. Survival times were compared using Kaplan–Meier curves and the log-rank test from GraphPad Prism version 4.0, GraphPad Software (San Diego, CA). The asterisks (* ) or (** ) indicate p < 0.05 or p < 0.01 respectively. PP, paraformaldehyde-killed B. pseudomallei. Data are from two independent experiments.

IFN-␥ level before infection correlated with some degree of protection (50% survival). In contrast, the serum IFN-␥ level of the PP + CpG ODN groups on day −5 was lower with less protective effects. On day 2 after infection, the PP + CpG ODN and PBS groups showed high levels of serum IFN-␥ whereas the CpG ODN and PP alone groups showed lower levels of IFN-␥ (Fig. 3B).

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(HK) as specific antigens for adaptive immunization. PP was found to confer higher percent protection than HK (Table 1 and Fig. 2). In silver stained SDS-PAGE gels, PP showed fewer protein bands when compared to HK (Fig. 1A). We found that a protein with molecular weight of 30 kDa in PP reacted with convalescent but not acute mouse sera and may therefore be possibly involved in inducing protective adaptive immune responses (Fig. 1B). It is unknown whether this 30 kDa protein is related to that of a previous study in which a 30 kDa protein was detected in the acute phase [33]. Surprisingly, we found no further protection in mice immunized with PP plus CpG ODN (Fig. 3A). In another study, active immunization with live attenuated B. pseudomallei combined with CpG dosing at different times prior to challenge gave enhanced protection [34]. In the PP group, we observed significantly increased levels of serum IFN-␥ on day −5, which coincided with protection (Fig. 3B). However, the high IFN-␥ production, although not significant, after B. pseudomallei infection in the PP + CpG ODN group indicates that some bacteria might still persist leading to higher cytokine production after challenge compared to PP alone (Fig. 3B). It is possible that the presence of high levels of cytokines after infection contributes to the pathology of melioidosis [6]. These results are reminiscent of other studies using CpG ODN and total soluble antigen of Toxoplasma gondii [35], or culture filtrate antigen of Mycobacterium tuberculosis [36] which enhanced IFN-␥ production and raised specific Th1 immune responses but failed to enhance the protective efficacy after challenge. In addition, the combination of CpG ODN with influenza M2e vaccine showed an enhanced Th1 immune response but failed to increase protection against virus challenge [37]. Hsieh et al. [36] showed no enhancement of protection when CpG ODN was added to culture filtrate antigen (CFP) of M. tuberculosis but in contrast, Freidag et al. showed enhanced protection when CpG ODN was added to BCG administered to BALB/c mice and challenged with aerosolized M. tuberculosis [38]. Hsieh et al. [36] suggested that the differences might be due to the antigen employed; in contrast to CFP, BCG persists intracellularly and induces a longer Th1 response. In summary, our study suggests that PP contains antigens that can stimulate protective responses in B. pseudomallei infection. However, percent survival was not increased when PP was used in combination with CpG ODN. Nevertheless, our finding that PP alone is protective against B. pseudomallei challenge justifies further investigations on killed bacteria for optimization as potential vaccine components. Further studies are necessary to clarify the underlying mechanisms and to investigate possible benefits for other protective candidate vaccines against B. pseudomallei infection.

4. Discussion Synthetic oligodeoxynucleotides containing unmethylated CpG dinucleotide motifs (CpG ODN) are capable of mimicking the immunostimulatory effects of bacterial DNA on B cells, monocytes/macrophages, NK cells and dendritic cells. After activation, B cells, monocytes/macrophages, NK cells and dendritic cells can secrete cytokines such as TNF-␣, IL-12 and type-1 IFNs to promote a Th1 immune response [22,29,30]. The effects of CpG ODN are therefore useful in clearing certain pathogens that are susceptible to a strong Th1 response. As examples, there are numerous reports showing that CpG ODN administration can protect mice from Listeria monocytogenes [25], Klebsiella pneumonia [24], Fransiella tularensis [31] and B. pseudomallei [26]. As shown in our previous study, the administration of CpG ODN to mice 2–10 days before B. pseudomallei challenge conferred better than 80–90% protection [26]. The protective effect of CpG-ODN on B. pseudomallei infection was extended to 30 days before infection by combination of CpG-ODN with cationic liposomes [32]. Since CpG ODN stimulates only innate immunity, we explored the use of paraformaldehyde-killed bacteria (PP) and heat-killed bacteria

Conflict of interest None. Acknowledgments This work was supported by the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program to A.P. and S.W. [Grant no. PHD/0007/2548]; Faculty of Medicine, Khon Kaen University, National Research University Project of Thailand, Office of the Higher Education Commission, through the Health Cluster (SHeP-GMS) and Melioidosis Research Center, Khon Kaen University, Thailand. References [1] Currie BJ, Fisher DA, Anstey NM, Jacups SP. Melioidosis: acute and chronic disease, relapse and re-activation. Trans R Soc Trop Med Hyg 2000;94:301–4. [2] Leelarasamee A, Bovornkitti S. Melioidosis: review and update. Rev Infect Dis 1989;11:413–25.

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Comparison of the protective effects of killed Burkholderia pseudomallei and CpG oligodeoxynucleotide against live challenge.

Melioidosis is a fatal disease caused by Burkholderia pseudomallei. Currently there is no vaccine available. Synthetic oligodeoxynucleotides with unme...
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