Progress toward development of vaccines against melioidosis: A review

Clinical Therapeutics/Volume 32, Number 8, 2010

Progress Toward Development of Vaccines Against Melioidosis: A Review Mitali Sarkar-Tyson, PhD1; and Richard W. Titball, PhD, DSc2 1Defence

Science and Technology Laboratory, Salisbury, United Kingdom; and 2School of Biosciences, University of Exeter, Exeter, United Kingdom ABSTRACT Background: Melioidosis is a serious and often fatal disease that is prevalent in subtropical and tropical climates, primarily in at-risk groups (eg, those with diabetes, alcoholism, or other cause of immunosuppression). Treatment is often unsuccessful, with infection frequently relapsing. Burkholderia pseudomallei, the etiologic agent of melioidosis, is inherently resistant to many antibiotics. Objective: This article reviews available evidence on the development of vaccines against melioidosis, including live attenuated vaccines, inactivated whole cell vaccines, and recombinant subunit vaccines. Methods: Web of Science and PubMed (1950–February 2010) were searched for relevant reports using the term Burkholderia pseudomallei alone and combined with live attenuated vaccine, inactivated vaccine, animal models, and immunity. The reference lists of identified articles were reviewed for additional relevant publications. Results: Studies in murine models suggest that protective immunity against B pseudomalleii may be induced by a range of living and nonliving immunogens. The strongest protective immunity was induced by live attenuated immunogens, although concerns about latency make it unlikely that such vaccines will be appropriate for use in humans. Heat-inactivated immunogens have shown promise, and several candidates for subunit vaccines have been tested. However, in all cases, it has been difficult to achieve induction of sterile immunity and protection against airborne infection. Conclusions: Live attenuated mutants of B pseudomallei have been found to be the most effective immunogens in mice, although it is unlikely that such mutants would be appropriate for a vaccine against melioidosis in humans. The ongoing challenge is to identify nonliving formulations that are able to induce good protective immunity. Both humoral and cell-mediated immunity are likely to be required. In this respect, naked DNA August 2010

vaccines have the potential to provide high-level protection. (Clin Ther. 2010;32:1437–1445) © 2010 Excerpta Medica Inc Key words: melioidosis, vaccines, live vaccines, inactivated vaccines, subunit vaccines.

INTRODUCTION Melioidosis is a disease prevalent in subtropical and tropical climates.1,2 In the Ubon Ratchathani province of northeast Thailand, for example, melioidosis is the most common cause of severe community-acquired septicemia, with a documented prevalence of 4.4 cases per 100,000.1 The reported mortality rate ranges from 19% in Australia to 50% in northeast Thailand.3 Although the disease also occurs in individuals with no obvious predisposition,4 melioidosis is most frequently seen in at-risk groups, including those with diabetes, alcoholism, or other causes of immunosuppression.1,5 In Australia and Thailand, for example, between 37% and 60% of patients with melioidosis have diabetes, whereas 39% of Australian patients with melioidosis are excessive users of alcohol.1 The worldwide incidence of melioidosis appears to be increasing, partly as a result of increased travel to endemic areas and partly because of increased reporting as a result of improved diagnostic tests.6 Nonetheless, given reports of the disease from a number of countries in which it was not previously found, the incidence of melioidosis is almost certainly underreported.7 The clinical diagnosis of melioidosis is complicated by its varying manifestations, which can range from acute fulminant sepsis to chronic illness.1,6,8 In some cases, a latent infection can reactivate years later to cause active disease.9 Accepted for publication July 8, 2010. doi:10.1016/j.clinthera.2010.07.020 0149-2918/$ - see front matter © 2010 Excerpta Medica Inc. All rights reserved.

1437

Clinical Therapeutics Melioidosis is caused by the gram-negative bacterium Burkholderia pseudomallei, which is inherently resistant to many antibiotics, including β-lactams, aminoglycosides, macrolides, and polymixins. Consequently, treatment may involve intensive and prolonged regimens that are often unsuccessful. The recurrence rate is reported to range from 13% to 26%,1,3 depending on the antibiotic used to treat the primary infection; 75% of these cases are a consequence of relapse rather than reinfection.1 There is currently no approved vaccine against melioidosis. This article reviews available data on the development of melioidosis vaccines, including live attenuated vaccines, inactivated whole cell vaccines, and recombinant subunit vaccines.

METHODS Web of Science and PubMed (1950–February 2010) were searched for relevant reports using the term Burkholderia pseudomalleii alone and combined with live attenuated vaccine, inactivated vaccine, animal models, and immunity. The reference lists of identified articles were reviewed for additional relevant publications.

RESULTS The literature search identified 1083 publications, of which 71 were included in the review. Of these, 12 involved animal models, 18 concerned protective immune responses, 9 examined live attenuated vaccines, 6 assessed inactivated whole cell vaccines, 20 studied B pseudomallei antigens as vaccine candidates, 3 involved DNA vaccines, and 3 involved polysaccharide conjugate vaccines.

activation and stimulates antibody production.11 This association between immunologic bias and resistance to infection suggests that macrophage activation by the Th1 response is important for combating melioidosis. The association of Th1 bias with resistance to infection is illustrated by the increased bactericidal activity of peritoneal exudate cell cultures containing macrophages from C57BL/6 mice compared with those containing macrophages from BALB/c mice.12 An inflammatory response is essential for clearance of B pseudomallei, and several investigators have reported increased expression of chemokines and cytokines in challenged mice.13–17 Investigation into the early immune response to infection in C57BL/6 mice compared with BALB/c mice revealed that the latter had higher concentrations of the 17 studied chemokines and cytokines.13 The presence of high levels of inflammatory proteins is not correlated with increased protection against B pseudomallei, as BALB/c mice are more susceptible to disease. It is possible that these chemokines and cytokines contribute to the pathology of meliodosis.13 The findings on the early immune response to infection in mice broadly correlate with observations in patients with melioidosis.18,19 A study of the immune response after exposure of mice to sublethal doses of B pseudomallei provided additional insight into the response to infection; granulocyte macrophage colonystimulating factor (GM-CSF) and the proapoptotic regulator Bad were found to be upregulated in BALB/c mice, whereas upregulation of cyclin-dependent kinase 5 (CDK5) was observed in C57BL/6 mice.20 GM-CSF has previously been found to play a role in immunity to bacterial infection21; however, the role of Bad and CDK5 is yet to be determined.

Animal Models A recent review discussed in detail the advantages and disadvantages of different animal models of B pseudomallei infection.10 Most studies evaluating pretreatments or therapies for melioidosis have involved murine models, in particular, BALB/c or C57BL/6 mice. BALB/c mice are highly susceptible to infection and are less able to resist infection than are resistant strains such as C57BL/6; the median lethal doses (MLDs) of B pseudomallei strain K96243 in BALB/c and C57BL/6 mice were 292 and 4 × 105 CFU, respectively.10 The differences in susceptibility may reflect differences in the immunologic bias of these mice. Th1 bias (eg, C57BL/6 mice) is important for macrophage activation, whereas Th2 bias (eg, BALB/c mice) inhibits macrophage 1438

Protective Immune Responses Understanding the relative roles of humoral and cellular immune responses in protection against B pseudomallei infection is essential to the design of an effective vaccine. There is no published information on the protective immune response to B pseudomallei in humans. However, individuals who have recovered from B pseudomallei infection exhibit antibodies to the bacterium, and the antibody titer has been reported to be broadly correlated with disease severity.4 It is likely that many of these antibodies are directed against cell-surface polysaccharides. The passive transfer of polyclonal sera against capsular polysaccharide or lipopolysaccharide (LPS) into naive mice was found to Volume 32 Number 8

M. Sarkar-Tyson and R.W. Titball double their survival time after subsequent challenge.22 Similar results have been reported in other animal models of infection. Diabetic rats were protected against B pseudomalleii infection by administration of polyclonal antibodies to LPS.23 In another study, passive transfer of monoclonal antibodies against capsular polysaccharide (3VEI5 and 4VA5) or LPS (CC6) protected mice against intraperitoneal challenge with 104 CFU of B pseudomallei strain NCTC4845 (~250 MLDs).24 Similar protection was reported with a monoclonal antibody directed against the capsular polysaccharide in an outbred strain of mice.25 Antibody to the polysaccharides has been reported to be opsonizing and to promote killing by polymorphonuclear leukocytes.26 The identity of protective antibodies directed against proteins is less well documented than the identity of those directed against polysaccharides, although sera from patients with melioidosis have been found to react to B pseudomalleii protein extracts,27 and a monoclonal antibody (4VH7) against an unidentified protein protected mice against B pseudomallei.24 Another study using a B pseudomallei protein microarray identified 170 antigens that reacted to sera from patients with melioidosis.28 Sterile immunity has rarely been reported, other than at very low challenge doses. Between 50% and 70% of mice immunized with killed bacterial cells were alive 45 days after intraperitoneal challenge with 40 MLDs of B pseudomallei, but bacteria could be isolated from 10 of 16 mice that were alive at the end of the study.29 Rather, it appears that antibodies are able to delay the onset of overt disease. For example, mice challenged with 5.4 × 103 CFU B pseudomallei strain NCTC4845 died by day 10, whereas groups of mice that had been passively immunized with monoclonal or polyclonal antibodies against LPS had mean times to death of 23.7 and 29.0 days, respectively, and showed no overt signs of disease until day 13.22 The inability of antibody to provide high-level protection against B pseudomallei may reflect the intracellular lifestyle of the organism, suggesting the importance of cell-mediated immunity for protection. Patients with severe melioidosis have been reported to have high concentrations of proinflammatory cytokines, immunoregulatory cytokines, and granzymes A and B (components of natural killer [NK]–cell and cytotoxic T-cell lytic granules), indicating a cellular immune response.30 A report of asymptomatic but seropositive individuals exhibiting stronger cellular adaptive responses compared with individuals with clinical August 2010

melioidosis further suggested a key role for cellular immunity.31 Experimental models of melioidosis in mice have begun to identify the mechanisms of immunity.32,33 Interferon-gamma (IFN-γ) has been found to be important for protection against acute sepsis in mice, and immunization with live immunogens was reported to activate antigen-specific CD4+ T cells that produced high levels of IFN-γ.34 NK cells and CD8+ T cells have also been found to produce IFN-γγ through a bystander pathway; although IFN-γγ protected mice from acute sepsis after infection with B pseudomallei, the animals did develop chronic disease.35 An analysis of the cell-mediated response in humans living in northeast Thailand reported evidence of T-cell priming,36 reflecting observations in the murine model of infection. The extent to which CD4+ T cells secreting IFN-γγ were activated was found to be correlated with individuals’ serologic status. CD8+ T cells and NK cells are also potential sources of IFN-γ in humans, which may explain the lack of correlation between HIV infection (in which numbers of CD4+ T cells would be reduced) and susceptibility to melioidosis.36 The immunologic responses to acute infection suggest the importance of IFN-γ. However, as with antibody alone, sterile immunity is difficult to achieve through IFN-γ alone. Taken together, the results of the foregoing studies suggest that a multicomponent vaccine may be required for protection against B pseudomallei.

Live Attenuated Vaccines Live attenuated vaccines are currently used in humans to induce protective immunity against disease caused by a number of bacterial pathogens, including Mycobacterium tuberculosis and Salmonella enterica serovar Typhi.37,38 The effectiveness of live attenuated vaccines depends both on the number of different antigens presented and on the ability to stimulate both the antibody and cell-mediated arms of the immune system. There are several reports of mouse studies in which attenuated mutants were able to induce protective immunity against challenge with virulent B pseudomallei (Table I).39–44 Because of the different immunization routes and mouse strains used, it is difficult to make meaningful comparisons of the relative abilities of these mutants to induce protective immunity. In a study of protection using an ilvII mutant, BALB/c mice eventually succumbed to infection,39 whereas C57BL/6 mice immunized with an aroC C mutant appeared to develop 1439

Clinical Therapeutics

Table I. Live attenuated mutants that have been reported to induce protective immunity against Burkholderia pseudomalleii infection in mice.

Gene ilvI39

serC C40 aroB41

purN42 purM42 BPSS150942 lipB42 pabB42 BipD43 aroC C44

Pathway/Gene Function Branched chain amino acid biosynthesis Serine biosynthesis Aromatic amino acid biosynthesis Purine biosynthesis Purine biosynthesis Hypothetical protein Lipoate P-aminobenzoate biosynthesis Type III secretion Aromatic amino acid biosynthesis

MLD, CFU 2.6 × 106

3.25 × 106 >1 × 106

106 –<107 >107 2 × 103–<107 2 × 102–<5 × 103 <107 NR NR

MLD = median lethal dose; NR = not reported.

sterile immunity.44 It is possible that this difference in protection reflected the differences between these strains of mice. The likelihood of an effective live attenuated vaccine against B pseudomallei being developed may be limited by the bacterium’s potential to establish a persistent infection. Successful development of such vaccines may depend on gaining a fuller understanding of the genetic basis underlying latency and how it can be effectively disabled.

Inactivated Whole Cell Vaccines Inactivated whole cell vaccines have been used successfully against a number of bacterial diseases, including typhoid fever and whooping cough.45 Administered 1440

on various immunization schedules, heat-inactivated B pseudomalleii immunogens have been found to provide some protection in the mouse model of infection.29,46,47 The results of these studies have been conflicting (Table II). One of the first published studies found that mice immunized with killed whole cells were completely protected for 7 days after challenge compared with the control group, which had no survivors.47 Longer-term survival data were not reported. In another study, 60%, 70%, and 60% of mice immunized with killed B pseudomallei, killed Burkholderia mallei, and killed Burkholderia thailandensis cells, respectively, survived 45 days after challenge with B pseudomallei strain K96243 at 40 times the MLD.29 In contrast, another group of investigators found that immunization with killed cells did not protect mice after B pseudomalleii challenge; the mice in this study were challenged by the intravenous route, which leads to acute infections similar to those produced by inhalational or aerosol challenge, possibly explaining the lack of protection.46 The contrasting results also may reflect differences in the strain of B pseudomalleii used for the preparation of killed cells, the dosing regimen, and/or the amount of immunogen used. Together, the foregoing results provide evidence that immunization with killed whole cell vaccines can provide protection against subsequent challenge with B pseudomallei in mice. However, sterile immunity is difficult to achieve. In 3 separate experiments, 0%, 20%, and 22.5% of the challenged animals exhibited a delayed time to death, and of 16 mice alive at the end of the study (day 45), 62.5% had evidence of bacterial colonization of organs post mortem.29 One approach to enhancing the protective response induced by inactivated preparations may be to combine them with immune adjuvants such as CpG, which promotes the production of Th1 and proinflammatory cytokines and the maturation/activation of professional antigen-presenting cells, activities that can accelerate and boost antigen-specific immune responses.48 This approach was successful when animals were immunized with dendritic cells pulsed with heat-killed B pseudomallei codelivered with the immune stimulator CpG 1826.49 Immunized animals were 60% to 70% protected, compared with 0% of naive controls, and crossprotection against a number of different B pseudomallei strains was observed. Immunized animals that were alive at the end of the experiment were clear of obvious infection, suggesting that this formulation may have induced sterile immunity. Further investigations are warVolume 32 Number 8

M. Sarkar-Tyson and R.W. Titball

Table II. Levels of protection against Burkholderia pseudomalleii provided by inactivated whole cell immunogens.

Sarkar-Tyson et al29

Variable Source of immunogen

Razak et al47 B pseudomallei strain not specified

B pseudomallei strain K96243

B pseudomallei strain K96243

B pseudomallei strain NCTC13179

1 × 108

1 × 108

1 × 108

290

5 × 105

Heat

Heat

Heat

Heat

Heat

BALB/c mice

BALB/c mice

BALB/c mice

BALB/c mice

Immunization route

Intraperitoneal

Intraperitoneal

Intraperitoneal

Subcutaneous

Intraperitoneal

Challenge strain

B pseudomallei strain K96243

B pseudomallei strain K96243

B pseudomallei strain K96243

B pseudomallei strain NCTC19178

B pseudomallei strain not specified

Challenge dose

4 × 104 CFU

4 × 104 CFU

92 CFU

20 CFU

Challenge route

Intraperitoneal

Intraperitoneal

Aerosol

Duration of study, d

35

35

35

40

7

Survival at end of experiment, %

50

60

0

0

100

Immunization dose, CFU Method of inactivation Animal model

B pseudomallei strain 576

Barnes and Ketheesan46

ranted to evaluate killed cells administered with other adjuvants.

B pseudomalleii Antigens as Vaccine Candidates Two criteria have been proposed for microbacterial proteins that have the potential to act as protective antigens. First, these proteins should be immunogenic and, ideally, should have been recognized by the immune system during infection.50 In practice, the reaction of antibody in convalescent sera to the protein is taken to indicate the immune system’s recognition of the protein. Proteins that evoke protective cellular responses but not humoral immunity would not be identified by this approach. Second, and not necessarily to the exclusion of the first criterion, the protein of interest should play a role in virulence. There is evidence that the capsular polysaccharide (type I O-PS51) and LPS (type II O-PS52) produced by B pseudomallei may be protective antigens and important determinants of virulence. The survival of individuAugust 2010

Intravenous

Mouse strain not specified

2 × 106 cells/ mL Intraperitoneal

als with melioidosis has been reported to be correlated with levels of serum antibody to LPS, further suggesting the value of this immunogen as a protective antigen.53 Immunization with capsular polysaccharide or LPS provided protection against subsequent challenge with B pseudomallei in mice,22 and passive immunization with antibodies against capsular polysaccharide or LPS reduced the lethality of infection in mice and diabetic rats.24,54 Although capsular polysaccharide and LPS show promise as vaccine candidates, immunization with these antigens alone does not appear to provide complete protection against disease or sterile immunity. Other polysaccharides encoded or expressed by B pseudomallei,53,55,56 including the exopolysaccharide,57–59 have yet to be evaluated as protective antigens. Patients who have recovered from melioidosis have antibodies that react with B pseudomallei proteins.36,42 A number of approaches to identifying these immunogenic proteins have been tested, including immunoscreening of expression libraries of B pseudomalleii genes, 1441

Clinical Therapeutics identification of antibody-reactive proteins in preparations enriched for surface-located antigens,27,60 and use of proteome arrays to identify immunogenic proteins.28 Some of these proteins have been tested for their ability to induce protective immunity in murine models. The most promising vaccine candidate identified to date among B pseudomallei proteins is the lipoproteinreleasing system transmembrane protein (LolC), an adenosine triphosphate–binding cassette transporter protein. Immunization of BALB/c mice with LolC and adjuvant immunostimulating complex (ISCOMS) and CpG oligodeoxynucleotide 10103 significantly protected the animals from infection after administration of B pseudomalleii K96243 at 70 times the MLD; at 13 days after challenge, 66% of the immunized group survived, compared with no survivors in the control group (P = 0.024).61 The same study investigated the protective effects of the adjuvants CpG, ISCOMS, monophosphoryl lipid A + trehalose docorynomycolate, and Emulsigen, alone and in combination. The only combination that provided significant protection was LolC delivered with ISCOMS and CpG oligodeoxynucleotide 10103 (P = 0.019). Further studies on the delivery of protective antigens is essential for the production of a subunit vaccine. More recently, 2 outer membrane proteins (OmpAs) of B pseudomallei that reacted with melioidosis convalescent sera have been identified: BPSL2522 and BPSL2765.62 The immunization of mice with either protein protected 50% of mice challenged with B pseudomallei strain D283 at 10 MLDs. However, all surviving mice had evidence of bacterial colonization of the spleen, indicating the failure of immunization to provide sterile immunity. The results of these studies indicate the potential for induction of protective immunity after immunization with individual B pseudomallei antigens. However, it is clear that a single component vaccine, at least delivered as a purified polysaccharide or purified protein, will not provide complete protection against disease. Two approaches to improving the efficacy of subunit vaccine candidates have been proposed: delivery as naked DNA vaccines or delivery as polysaccharide conjugate vaccines.

DNA Vaccines DNA vaccines are potent stimulators of both humoral and cell-mediated immunity. Candidate DNA vaccines against melioidosis have been investigated based on the 1442

ability to promote cell-mediated immunity. Almost all published studies have used DNA vaccines encoding the B pseudomallei flagellar subunit gene, fliC.63,64 The immunization of mice with fliC naked DNA resulted in the induction of both antibody and cell-mediated responses, and immunized mice that were challenged with wild-type B pseudomallei had reduced numbers of bacteria in their livers and spleens compared with control animals.64 The potency of this naked DNA construct was improved by incorporating CpG oligodeoxynucleotide motifs into the DNA plasmid/fli / C construct; the mean survival rate increased from 80% in the DNA plasmid/fli / C–immunized group to 93.3% in the DNA plasmid/CpG-fliC–immunized group.63 Based on the available information, it is not yet possible to assess the overall potential of naked DNA as a delivery system for B pseudomallei vaccine antigens. Delivery of fliC in this way has provided only modest levels of protection, and there have been no reports on the efficacy of lolC C or ompA naked DNA vaccines as protective antigens.

Polysaccharide Conjugate Vaccines The immunogenicity of bacterial polysaccharides can be improved by covalently linking them to protein carriers to form a conjugate.65 Polysaccharide–protein glycoconjugates work by promoting T-cell–dependent immune responses, thus enhancing immunologic memory.66 Polysaccharide conjugate vaccines have been used successfully to protect against a number of pathogens. One published study tested a conjugate vaccine (LPS conjugated to flagellin) against B pseudomallei challenge.23 Antisera raised against the conjugate were found to be protective in diabetic rats, but there are no reports of active immunization with the conjugate.

DISCUSSION It seems likely that humans are capable of developing protective immunity against B pseudomalleii infection, as many individuals living in areas where the bacterium is present in the soil do not develop disease.36,677 In addition, there is evidence that some individuals become infected but do not develop overt disease until they later develop a condition such as diabetes.9,68 However, the explanation for this protective immunity remains elusive. A wide range of studies in mice have indicated the potential to induce protective immunity after dosing with live, killed, or subunit immunogens. However, generation of sterile immunity and protection against Volume 32 Number 8

M. Sarkar-Tyson and R.W. Titball airborne challenge are difficult to achieve. This may reflect the intracellular lifestyle of the organism and the associated ability to evade the immune system. In addition, it is not clear to what extent findings in mice can be used to predict the likely responses to immunogens in humans; this is further complicated by the fact that different strains of mice have been found to differ markedly in their susceptibility to infection. It seems likely that meaningful extrapolation from mice to humans will depend on a more detailed understanding of the nature of the protective immune responses in mice and a clearer understanding of the reasons for the differing susceptibilities of mouse strains to infection.

CONCLUSIONS Live attenuated mutants of B pseudomallei have been found to be the most effective immunogens in mice. However, it is unlikely that live attenuated mutants will be appropriate for use in humans. The ongoing challenge is to identify nonliving formulations that are able to induce good protective immunity. Both humoral and cell-mediated immunity are likely to be required. In this respect, naked DNA vaccines have the potential to provide high-level protection.

ACKNOWLEDGMENT The authors have indicated that they have no conflicts of interest with regard to the content of this article.

REFERENCES 1. Cheng AC, Currie BJ. Melioidosis: Epidemiology, pathophysiology, and management [published correction appears in Clin Microbiol Rev. 2007;20:533]. Clin Microbiol Rev. 2005; 18:383–416. 2. Dance DA. Melioidosis: The tip of the iceberg? Clin Microbiol Rev. 1991;4:52–60. 3. Wuthiekanun V, Peacock SJ. Management of melioidosis. Expert Rev Anti Infect Ther. 2006;4:445–455. 4. Wiersinga WJ, van der Poll T, White NJ, et al. Melioidosis: Insights into the pathogenicity of Burkholderia pseudomallei. Nat Rev Microbiol. 2006;4:272–282. 5. Suputtamongkol Y, Chaowagul W, Chetchotisadk P, et al. Risk factors for melioidosis and bacteremic melioidosis. Clin Infect Dis. 1999;29:408–413. 6. White NJ. Melioidosis. Lancet. 2003;361:1715–1722. 7. Currie BJ, Dance DA, Cheng AC. The global distribution of Burkholderia pseudomalleii and melioidosis: An update. Trans R Soc Trop Med Hyg. 2008;102(Suppl 1):S1–S4. 8. Currie BJ. Melioidosis: An important cause of pneumonia in residents of and travellers returned from endemic regions. Eur Respir J. 2003;22:542–550.

August 2010

9. Ngauy V, Lemeshev Y, Sadkowski L, Crawford G. Cutaneous melioidosis in a man who was taken as a prisoner of war by the Japanese during World War II. J Clin Microbiol. 2005;43:970–972. 10. Titball RW, Russell P, Cuccui J, et al. Burkholderia pseudomallei: Animal models of infection. Trans R Soc Trop Med Hyg. 2008;102(Suppl 1):S111–S116. 11. Mills CD, Kincaid K, Alt JM, et al. M-1/M-2 macrophages and the Th1/Th2 paradigm. J Immunol. 2000;164:6166– 6173. 12. Leakey AK, Ulett GC, Hirst RG. BALB/c and C57BI/6 mice infected with virulent Burkholderia pseudomalleii provide contrasting animal models for the acute and chronic forms of human melioidosis. Microb Pathog. 1998;24:269–275. 13. Tan GY, Liu Y, Sivalingam SP, et al. Burkholderia pseudomallei aerosol infection results in differential inflammatory responses in BALB/c and C57BI/6 mice. J Med Microbiol. 2008;57:508–515. 14. Ulett GC, Ketheesan N, Hirst RG. Cytokine gene expression in innately susceptable BALB/c mice and relatively resistant C57BL/6 mice during infection with virulent Burkholderia pseudomallei. Infect Immun. 2000;68:2034–2042. 15. Ulett GC, Ketheesan N, Hirst RG. Proinflammatory cytokine mRNA responses in experimental Burkholderia pseudomalleii infection in mice. Acta Trop. 2000;74:229–234. 16. Wiersinga WJ, Dessing MC, van der Poll T. Gene-expression profiles in murine melioidosis. Microbes Infect. 2008;10: 868–877. 17. Wiersinga WJ, de Vos AF, de Beer R, et al. Inflammation patterns induced by different Burkholderia species in mice. Cell Microbiol. 2008;10:81–87. 18. Brown AE, Dance DA, Suputtamongkol Y, et al. Immune cell activation in melioidosis: Increased serum levels of interferon-gamma and soluble interleukin-2 receptors without change in soluble CD8 protein. J Infect Dis. 1991;163: 1145–1148. 19. Suputtamongkol Y, Kwiatkowski D, Dance DA, et al. Tumor necrosis factor in septicemic melioidosis. J Infect Dis. 1992;165:561–564. 20. Ulett GC, Labrooy JT, Currie BJ, et al. A model of immunity to Burkholderia pseudomallei: Unique responses following immunization and acute lethal infection. Microbes Infect. 2005;7:1263–1275. 21. LeVine AM, Reed JA, Kurak KE, et al. GM-CSF-deficient mice are susceptible to pulmonary group B streptococcal infection. J Clin Invest. 1999;103:563–569. 22. Nelson M, Prior JL, Lever MS, et al. Evaluation of lipopolysaccharide and capsular polysaccharide as subunit vaccines against experimental melioidosis. J Med Microbiol. 2004;53:1177–1182. 23. Brett PJ, Woods DE. Structural and immunological characterization of Burkholderia pseudomalleii O-polysaccharideflagellin protein conjugates. Infect Immun. 1996;64:2824– 2828.

1443

Clinical Therapeutics 24. Jones SM, Ellis JF, Russell P, et al. Passive protection against Burkholderia pseudomalleii infection in mice by monoclonal antibodies against capsular polysaccharide, lipopolysaccharide or proteins. J Med Microbiol. 2002;51:1055–1062. 25. Bottex C, Gauthier YP, Hagen RM, et al. Attempted passive prophylaxis with a monoclonal anti-Burkholderia pseudomalleii exopolysaccharide antibody in a murine model of melioidosis. Immunopharmacol Immunotoxicol. 2005;27:565–583. 26. Ho M, Schollaardt T, Smith MD, et al. Specificity and functional activity of anti-Burkholderia pseudomalleii polysaccharide antibodies. Infect Immun. 1997;65:3648–3653. 27. Harding SV, Sarkar-Tyson M, Smither SJ, et al. The identification of surface proteins of Burkholderia pseudomallei. Vaccine. 2007;25:2664–2672. 28. Felgner PL, Kayala MA, Vigil A, et al. A Burkholderia pseudomalleii protein microarray reveals serodiagnostic and cross-reactive antigens. Proc Natl Acad Sci U S A. 2009;106:13499–13504. 29. Sarkar-Tyson M, Smither SJ, Harding SV, et al. Protective efficacy of heat-inactivated B. thailandensis, B. malleii or B. pseudomalleii against experimental melioidosis and glanders. Vaccine. 2009;27:4447–4451. 30. Lauw FN, Simpson AJ, Hack CE, et al. Soluble granzymes are released during human endotoxemia and in patients with severe infection due to gram-negative bacteria. J Infect Dis. 2000;182:206–213. 31. Barnes JL, Warner J, Melrose W, et al. Adaptive immunity in melioidosis: A possible role for T cells in determining outcome of infection with Burkholderia pseudomallei. Clin Immunol. 2004;113:22–28. 32. Haque A, Easton A, Smith D, et al. Role of T cells in innate and adaptive immunity against murine Burkholderia pseudomalleii infection. J Infect Dis. 2006;193:370–379. 33. Santanirand P, Harley VS, Dance DA, et al. Obligatory role of gamma

1444

34.

35.

36.

37.

38.

39.

40.

41.

42.

interferon for host survival in a murine model of infection with Burkholderia pseudomallei. Infect Immun. 1999;67:3593–3600. Haque A, Chu K, Easton A, et al. A live experimental vaccine against Burkholderia pseudomalleii elicits CD4+ T cell-mediated immunity, priming T cells specific for 2 type III secretion system proteins. J Infect Dis. 2006;194:1241–1248. Lertmemongkolchai G, Cai G, Hunter CA, Bancroft GJ. Bystander activation of CD8+ T cells contributes to the rapid production of IFN-gamma in response to bacterial pathogens. J Immunol. 2001;166:1097–1105. Tippayawat P, Saenwongsa W, Mahawantung J, et al. Phenotypic and functional characterization of human memory T cell responses to Burkholderia pseudomallei. PLoS Negl Trop Dis. 2009;3:e407. Arya SC, Agarwal N. Re: Typhoid fever vaccines: Systematic review and meta-analysis of randomised clinical trials [comment]. Vaccine. 2008;26: 291. Hawkridge A. Clinical studies of TB vaccines. Hum Vaccin. 2009;5:773– 776. Atkins T, Prior P, Mack K, et al. Characterisation of an acapsular mutant of Burkholderia pseudomallei identified by signature tagged mutagenesis. J Med Microbiol. 2002;51: 539–547. Rodrigues F, Sarkar-Tyson M, Harding SV, et al. Global map of growthregulated gene expression in Burkholderia pseudomallei, the causative agent of melioidosis. J Bacteriol. 2006; 188:8178–8188. Cuccui J, Easton A, Chu KK, et al. Development of signature-tagged mutagenesis in Burkholderia pseudomalleii to identify genes important in survival and pathogenesis. Infect Immun. 2007;75:1186–1195. Breitbach K, Köhler J, Steinmetz I. Induction of protective immunity against Burkholderia pseudomalleii using attenuated mutants with defects in

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

the intracellular life cycle. Trans R Soc Trop Med Hyg. 2008;102(Suppl 1): S89–S94. Stevens MP, Haque A, Atkins T, et al. Attenuated virulence and protective efficacy of a Burkholderia pseudomallei bsa type III secretion mutant in murine models of melioidosis. Microbiology. 2004;150:2669–2676. Srilunchang T, Proungvitaya T, Wongratanacheewin S, et al. Construction and characterization of an unmarked aroC deletion mutant of Burkholderia pseudomalleii strain A2. Southeast Asian J Trop Med Public Health. 2009;40:123–130. Plotkin SA. Vaccines: The fourth century. Clin Vaccine Immunol. 2009; 16:1709–1719. Barnes JL, Ketheesan N. Development of protective immunity in a murine model of melioidosis is influenced by the source of Burkholderia pseudomalleii antigens. Immunol Cell Biol. 2007;85:551–557. Razak CE, Ismail G, Embim N, Omar O. Protection studies using whole cells and partially purified toxic material (PPTM) of Pseudomonas pseudomallei. Malays Appl Biol. 1986;15:105–111. Klinman DM. Adjuvant activity of CpG oligodeoxynucleotides. Int Rev Immunol. 2006;25:135–154. Elvin SJ, Healey GD, Westwood A, et al. Protection against heterologous Burkholderia pseudomalleii strains by dendritic cell immunization. Infect Immun. 2006;74:1706–1711. Bambini S, Rappuoli R. The use of genomics in microbial vaccine development. Drug Discov Today. 2009;14:252–260. Reckseidler S, DeShazer D, Sokol PA, Woods DE. Detection of bacterial virulence genes by subtractive hybridization: Identification of capsular polysaccharide of Burkholderia pseudomalleii as a major virulence determinant. Infect Immun. 2001;69: 34–44. DeShazer D, Brett PJ, Woods DE. The type II O-antigenic polysaccha-

Volume 32 Number 8

M. Sarkar-Tyson and R.W. Titball

53.

54.

55.

56.

57.

58.

59.

60.

61.

62.

ride moiety of Burkholderia pseudomalleii lipopolysaccharide is required for serum resistance and virulence. Mol Microbiol. 1998;30:1081–1100. Sarkar-Tyson M, Thwaite JE, Harding SV, et al. Polysaccharides and virulence of Burkholderia pseudomallei. J Med Microbiol. 2007;56:1005–1010. Bryan LE, Wong S, Woods DE, et al. Passive protection of diabetic rats with antisera specific for the polysaccharide portion of the lipopolysaccharide isolated from Pseudomonas pseudomallei. Can J Infect Dis. 1994;5:170–178. Holden MT, Titball RW, Peacock SJ, et al. Genomic plasticity of the causative agent of melioidosis, Burkholderia pseudomallei. Proc Natl Acad Sci U S A. 2004;101:14240–14245. Reckseidler-Zenteno SL, Moore R, Woods DE. Genetics and function of the capsules of Burkholderia pseudomalleii and their potential as therapeutic targets. Mini Rev Med Chem. 2009;9:265–271. Nimtz M, Wray V, Domke T, et al. Structure of an acidic exopolysaccharide of Burkholderia pseudomallei. Eur J Biochem. 1997;250:608–616. Steinmetz I, Rohde M, Brenneke B. Purification and characterization of an exopolysaccharide of Burkholderia (Pseudomonas) pseudomallei. Infect Immun. 1995;63:3959–3965. Steinmetz I, Nimtz M, Wray V, et al. Exopolysaccharides of Burkholderia pseudomallei. Acta Trop. 2000;74: 211–214. Jitsurong S, Chirathaworn C, Brown NF, et al. Searching for virulence Burkholderia pseudomallei genes by immunoscreening a lambda ZAPII expressed genomic library. Southeast Asian J Trop Med Public Health. 2003;34:810–821. Harland DN, Chu K, Haque A, et al. Identification of a LolC homologue in Burkholderia pseudomallei, a novel protective antigen for melioidosis. Infect Immun. 2007;75:4173–4180. Hara Y, Mohamed R, Nathan S. Immunogenic Burkholderia pseudomallei

August 2010

outer membrane proteins as potential candidate vaccine targets. PLoS One. 2009;4:e6496. 63. Chen YS, Hsiao YS, Lin HH, et al. Immunogenicity and anti-Burkholderia pseudomalleii activity in Balb/c mice immunized with plasmid DNA encoding flagellin. Vaccine. 2006;24: 750–758. 64. Chen YS, Hsiao YS, Lin HH, et al. Immunogenicity and anti-Burkholderia pseudomallei activity in Balb/c mice immunized with plasmid DNA encoding flagellin. Vaccine. 2006;24:750–758. 65. Jones C. Vaccines based on the cell surface carbohydrates of pathogenic bacteria. An Acad Bras Cienc. 2005; 77:293–324.

66. Wuorimaa T, Käyhty H, Eskola J, et al. Activation of cell-mediated immunity following immunization with pneumococcal conjugate or polysaccharide vaccine. Scand J Immunol. 2001;53:422–428. 67. Charoenwong P, Lumbiganon P, Puapermpoonsiri S. The prevalence of the indirect hemagglutination test for melioidosis in children in an endemic area. Southeast Asian J Trop Med Public Health. 1992;23:698– 701. 68. Mackowiak PA, Smith JW. Septicemic melioidosis. Occurrence following acute influenza A six years after exposure in Vietnam. JAMA. 1978; 240:764–766.

Address correspondence to: Mitali Sarkar-Tyson, Defence Science and Technology Laboratory, Porton Down, Salisbury, Wiltshire SP4 0JQ, United Kingdom. E-mail: [email protected] 1445