Development of a sensitive competitive enzyme-linked immunosorbent assay for serodiagnosis of Burkholderia mallei, a Tier 1 select agent.

Glanders is a highly contagious and potentially serious disease caused by Burkholderia mallei, a Tier 1 select agent. In this study, we raised a monoclonal antibody (mAb) against the lipopolysaccharide (LPS) of B. mallei and developed a competitive enzyme-linked immunosorbent assay (cELISA) for B. mallei infection. Using the titrated optimal conditions of B. mallei-LPS (2 ng) for microtiter plate coating, sample serum dilution at 1:20 and 3.5 ng/µL anti-LPS mAb B5, the cutoff value of the cELISA was determined using serum samples from 136 glanders-free seronegative horses in Hong Kong. All calculated percentage inhibition (PI) values from these seronegative samples were below 39.6% inhibition (1.5 standard deviations above mean PI) and was used as the cutoff value. The diagnostic sensitivity of the developed LPS-based cELISA was first evaluated using sera from donkeys and mice inoculated with B. mallei. An increasing trend of PI values above the defined cELISA cutoff observed in the donkey and mouse sera suggested positive detection of anti-LPS antibodies. The sensitivity and specificity of the LPS-based cELISA was further evaluated using 31 serologically positive horse sera from glanders outbreaks in Bahrain and Kuwait, of which 30 were tested positive by the cELISA; and 21 seronegative horse sera and 20 seronegative donkey sera from Dubai, of which all were tested negative by the cELISA. A cELISA with high sensitivity (97.2%) and specificity (100%) for the detection of B. mallei antibodies in different animals was developed.

Pathological and Immunohistochemical Analyses of Naturally Occurring Equine Glanders Using an Anti-BpaB Antibody.

Glanders is caused by the gram-negative bacterium Burkholderia mallei. In this study, we investigated the histopathology and immunohistochemical localization of B. mallei in natural cases of equine glanders. Four horses showing clinical signs of nasal discharge and multiple cutaneous nodules or papulae in the hindlimbs and abdomen were reported in Mongolia. They tested positive for B. mallei infection on complement fixation, Rose Bengal agglutination, and mallein tests. Gross and histological lesions observed in these cases were similar to those previously reported in equine glanders. Immunohistochemistry using a monoclonal antibody to B. mallei BpaB showed localization of the bacterial antigen in the cytoplasm of neutrophils, macrophages, epithelioid cells, and multinucleated giant cells in the pyogranulomas and abscesses in target organs. Some alveolar type II cells and bronchiolar epithelial cells also contained the antigen. These results suggest that the anti-BpaB antibody is useful for identifying B. mallei-infected cell types in naturally infected horses.

The Peptidoglycan-associated lipoprotein Pal contributes to the virulence of Burkholderia mallei and provides protection against lethal aerosol challenge.

BURKHOLDERIA MALLEI: is a highly pathogenic bacterium that causes the fatal zoonosis glanders. The organism specifies multiple membrane proteins, which represent prime targets for the development of countermeasures given their location at the host-pathogen interface. We investigated one of these proteins, Pal, and discovered that it is involved in the ability of B. mallei to resist complement-mediated killing and replicate inside host cells in vitro, is expressed in vivo and induces antibodies during the course of infection, and contributes to virulence in a mouse model of aerosol infection. A mutant in the pal gene of the B. mallei wild-type strain ATCC 23344 was found to be especially attenuated, as BALB/c mice challenged with the equivalent of 5,350 LD50 completely cleared infection. Based on these findings, we tested the hypothesis that a vaccine containing the Pal protein elicits protective immunity against aerosol challenge. To achieve this, the pal gene was cloned in the vaccine vector Parainfluenza Virus 5 (PIV5) and mice immunized with the virus were infected with a lethal dose of B. mallei. These experiments revealed that a single dose of PIV5 expressing Pal provided 80% survival over a period of 40 days post-challenge. In contrast, only 10% of mice vaccinated with a PIV5 control virus construct survived infection. Taken together, our data establish that the Peptidoglycan-associated lipoprotein Pal is a critical virulence determinant of B. mallei and effective target for developing a glanders vaccine.

Serological Survey of Humans Exposed to Burkholderia mallei-Infected Equids: A Public Health Approach.

Glanders is a fatal bacterial infection of equids caused by Burkholderia mallei. The infection can be transmitted to humans through prolonged direct contact with glanderous equids. Recently, reemergence of equine glanders has been reported in many countries. To investigate zoonotic transmission of B mallei infection, sera were collected from 538 humans including equine handlers and veterinary professionals exposed to glanderous equids. Samples were tested by ELISA (enzyme-linked immunosorbent assay) and complement fixation test and found negative for B mallei-specific antibodies. Even though there was no incidence of human glanders during this survey period, occupational exposure will continue to remain a serious concern and a key risk factor. Therefore, we emphasize the need for intersectoral collaboration and coordination among veterinary, human, and public health authorities for continuous surveillance and monitoring of human glanders under one health concept.

Development of a microsphere-based immunoassay for the serological detection of glanders in equids.

Burkholderia mallei is the etiologic agent of glanders, an infectious disease of solipeds, with renewed scientific interest due to its increasing incidence in different parts of the world. More rapid, sensitive and specific assays are required by laboratories for confirmatory testing of this disease. A microsphere-based immunoassay consisting of beads coated with B. mallei recombinant proteins (BimA, GroEL, Hcp1, and TssB) has been developed for the serological diagnosis of glanders. The proteins' performance was compared with the OIE reference complement fixation test (CFT) and an indirect enzyme-linked immunosorbent assay (iELISA) on a large panel of sera comprised of uninfected horses (n=198) and clinically confirmed cases of glanders from India and Pakistan (n=99). Using Receiver Operating Characteristics (ROC) analysis and adjusting the cutoff levels, Hcp1 (Se=100%, Sp=99.5%) and GroEL (Se= 97%, Sp=99.5%) antigens exhibited the best specificity and sensitivity. Neither Hcp1 and GroEL proteins, nor iELISA reacted with doubtful and positive CFT samples from glanders free countries which further confirmed the false positive reactions seen in CFT.

Lipopolysaccharides from Different Burkholderia Species with Different Lipid A Structures Induce Toll-Like Receptor 4 Activation and React with Melioidosis Patient Sera.

Lipopolysaccharides (LPSs) of Gram-negative bacteria comprise lipid A, core, and O-polysaccharide (OPS) components. Studies have demonstrated that LPSs isolated from the pathogenic species Burkholderia pseudomallei and Burkholderia mallei and from less-pathogenic species, such as Burkholderia thailandensis, are potent immune stimulators. The LPS structure of B. pseudomallei, the causative agent of melioidosis, is highly conserved in isolates from Thailand; however, the LPSs isolated from other, related species have not been characterized to enable understanding of their immune recognition and antigenicities. Here, we describe the structural and immunological characteristics of the LPSs isolated from eight Burkholderia species and compare those for B. pseudomallei to those for the other seven species. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), gas chromatography (GC), SDS-PAGE, Toll-like receptor 4 (TLR4) stimulation, and immunoblot analysis were performed on these Burkholderia species. MALDI-TOF profiles demonstrated that Burkholderia lipid A contains predominantly penta-acylated species modified with 4-amino-4-deoxy-arabinose residues at both terminal phosphate groups. The lipid A could be differentiated based on mass differences at m/z 1,511, 1,642, 1,773, and 1,926 and on fatty acid composition. LPSs of all species induced TLR4-dependent NF-κB responses; however, while SDS-PAGE analysis showed similar LPS ladder patterns for B. pseudomallei, B. thailandensis, and B. mallei, these patterns differed from those of other Burkholderia species. Interestingly, immunoblot analysis demonstrated that melioidosis patient sera cross-reacted with OPSs of other Burkholderia species. These findings can be used to better understand the characteristics of LPS in Burkholderia species, and they have implications for serological diagnostics based on the detection of antibodies to OPS.

Melioidosis patient serum-reactive synthetic tetrasaccharides bearing the predominant epitopes of Burkholderia pseudomallei and Burkholderia mallei O-antigens.

Melioidosis and glanders, respectively caused by the Gram-negative bacteria Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm), are considered as urgent public health issues in developing countries and potential bioterrorism agents. Bp and Bm lipopolysaccharides (LPS) have been identified as attractive vaccine candidates for the development of prophylactic measures against melioidosis and glanders. Bp and Bm express structurally similar LPSs wherein the O-antigen (OAg) portion consists of a heteropolymer whose repeating unit is a disaccharide composed of d-glucose and 6-deoxy-l-talose residues, the latter being diversely acetylated and methylated. Herein we report the synthesis of two tetrasaccharides mimicking the main substitution epitopes of Bp and Bm LPS OAgs. The assembly of the tetrasaccharides was achieved using a sequential glycosylation strategy while relying on the late-stage epimerization of the inner rhamnose into a 6-deoxy-l-talose residue. We show that these synthetic compounds strongly react with culture-confirmed Thai melioidosis patient serum and closely mimic the antigenicity of native Bp OAg. Our results suggest that these tetrasaccharides could be suitable candidates for the development of vaccines and/or diagnostic tools against melioidosis and glanders.

Evaluation of Burkholderia mallei ΔtonB Δhcp1 (CLH001) as a live attenuated vaccine in murine models of glanders and melioidosis.

BACKGROUND: Glanders caused by Burkholderia mallei is a re-emerging zoonotic disease affecting solipeds and humans. Furthermore, B. mallei is genetically related to B. pseudomallei, which is the causative agent of melioidosis. Both facultative intracellular bacteria are classified as tier 1 select biothreat agents. Our previous study with a B. mallei ΔtonB Δhcp1 (CLH001) live-attenuated vaccine demonstrated that it is attenuated, safe and protective against B. mallei wild-type strains in the susceptible BALB/c mouse model. METHODOLOGY/PRINCIPAL FINDING: In our current work, we evaluated the protective efficacy of CLH001 against glanders and melioidosis in the more disease-resistant C57BL/6 mouse strain. The humoral as well as cellular immune responses were also examined. We found that CLH001-immunized mice showed 100% survival against intranasal and aerosol challenge with B. mallei ATCC 23344. Moreover, this vaccine also afforded significant cross-protection against B. pseudomallei K96243, with low level bacterial burden detected in organs. Immunization with a prime and boost regimen of CLH001 induced significantly greater levels of total and subclasses of IgG, and generated antigen-specific splenocyte production of IFN-γ and IL-17A. Interestingly, protection induced by CLH001 is primarily dependent on humoral immunity, while CD4+ and CD8+ T cells played a less critical protective role. CONCLUSIONS/SIGNIFICANCE: Our data indicate that CLH001 serves as an effective live attenuated vaccine to prevent glanders and melioidosis. The quantity and quality of antibody responses as well as improving cell-mediated immune responses following vaccination need to be further investigated prior to advancement to preclinical studies.

Use of Immunohistochemistry to Demonstrate In Vivo Expression of the Burkholderia mallei Virulence Factor BpaB During Experimental Glanders.

Burkholderia mallei causes the highly contagious and debilitating zoonosis glanders, which infects via inhalation or percutaneous inoculation and often culminates in life-threatening pneumonia and sepsis. In humans, glanders is difficult to diagnose and requires prolonged antibiotic therapy with low success rates. No vaccine exists to protect against B. mallei, and there is concern regarding its use as a bioweapon. The authors previously identified the protein BpaB as a potential target for devising therapies due to its role in adherence to host cells and the formation of biofilms in vitro and its contribution to pathogenicity in a mouse model of glanders. In the present study, the authors developed an immunostaining approach to probe tissues of experimentally infected animals and demonstrated that BpaB is produced exclusively in vivo by wild-type B. mallei in target organs from mice and marmosets. They detected the expression of BpaB by B. mallei both extracellularly and within macrophages, neutrophils, and epithelial cells in respiratory tissues (7/10 marmoset; 2/2 mouse). The authors also noted the intracellular expression of BpaB by B. mallei in macrophages in the regional lymph nodes of mice (2/2 tissues) and MALT of marmosets (4/5 tissues). It is interesting that B. mallei bacteria infecting distal organs did not express BpaB (2/2 mice; 3/3 marmosets), suggesting that the protein is not necessary for bacterial fitness in these anatomic locations. These findings underscore the value of BpaB as a target for developing medical countermeasures and provide insight into its role in pathogenesis.

Identification of a new diagnostic antigen for glanders using immunoproteome analysis.

Glanders is a disease of horses, donkeys and mules. The causative agent Burkholderia mallei, is a biorisk group 3 pathogen and is also a biothreat agent. Simple and rapid diagnostic tool is essential for control of glanders. Using a proteomic approach and immunoblotting with equine sera, we identified 12 protein antigens that may have diagnostic potential. Various immunoreactive proteins e.g. GroEL, translation elongation factor Tu, elongation factor Ts, arginine deiminase, malate dehydrogenase, DNA directed RNA polymerase subunit alpha were identified on 2-dimentional immunoblots. One of these proteins, GroEL, was cloned and expressed in E. coli and purified using Ni-NTA affinity chromatography. The recombinant GroEL protein was evaluated in ELISA format on a panel of glanders positive (n=49) and negative (n=79) equine serum samples to determine its diagnostic potential. The developed ELISA had a sensitivity and specificity of 96 and 98.7% respectively. The results of this study highlight the potential of GroEL in serodiagnosis of glanders.

Antibodies against In Vivo-Expressed Antigens Are Sufficient To Protect against Lethal Aerosol Infection with Burkholderia mallei and Burkholderia pseudomallei.

Burkholderia mallei, a facultative intracellular bacterium and tier 1 biothreat, causes the fatal zoonotic disease glanders. The organism possesses multiple genes encoding autotransporter proteins, which represent important virulence factors and targets for developing countermeasures in pathogenic Gram-negative bacteria. In the present study, we investigated one of these autotransporters, BatA, and demonstrate that it displays lipolytic activity, aids in intracellular survival, is expressed in vivo, elicits production of antibodies during infection, and contributes to pathogenicity in a mouse aerosol challenge model. A mutation in the batA gene of wild-type strain ATCC 23344 was found to be particularly attenuating, as BALB/c mice infected with the equivalent of 80 median lethal doses cleared the organism. This finding prompted us to test the hypothesis that vaccination with the batA mutant strain elicits protective immunity against subsequent infection with wild-type bacteria. We discovered that not only does vaccination provide high levels of protection against lethal aerosol challenge with B. mallei ATCC 23344, it also protects against infection with multiple isolates of the closely related organism and causative agent of melioidosis, Burkholderia pseudomallei Passive-transfer experiments also revealed that the protective immunity afforded by vaccination with the batA mutant strain is predominantly mediated by IgG antibodies binding to antigens expressed exclusively in vivo Collectively, our data demonstrate that BatA is a target for developing medical countermeasures and that vaccination with a mutant lacking expression of the protein provides a platform to gain insights regarding mechanisms of protective immunity against B. mallei and B. pseudomallei, including antigen discovery.

Burkholderia pseudomallei and Burkholderia mallei vaccines: Are we close to clinical trials?

B. pseudomallei is the cause of melioidosis, a serious an often fatal disease of humans and animals. The closely related bacterium B. mallei, which cases glanders, is considered to be a clonal derivative of B. pseudomallei. Both B. pseudomallei and B. mallei were evaluated by the United States and the former USSR as potential bioweapons. Much of the effort to devise biodefence vaccines in the past decade has been directed towards the identification and formulation of sub-unit vaccines which could protect against both melioidosis and glanders. A wide range of proteins and polysaccharides have been identified which protective immunity in mice. In this review we highlight the significant progress that has been made in developing glycoconjugates as sub-unit vaccines. We also consider some of the important the criteria for licensing, including the suitability of the "animal rule" for assessing vaccine efficacy, the protection required from a vaccine and the how correlates of protection will be identified. Vaccines developed for biodefence purposes could also be used in regions of the world where naturally occurring disease is endemic.

Innate immune response to Burkholderia mallei.

PURPOSE OF REVIEW: Burkholderia mallei is a facultative intracellular pathogen that causes the highly contagious and often the fatal disease, glanders. With its high rate of infectivity via aerosol and recalcitrance toward antibiotics, this pathogen is considered a potential biological threat agent. This review focuses on the most recent literature highlighting host innate immune response to B. mallei. RECENT FINDINGS: Recent studies focused on elucidating host innate immune responses to the novel mechanisms and virulence factors employed by B. mallei for survival. Studies suggest that pathogen proteins manipulate various cellular processes, including host ubiquitination pathways, phagosomal escape, and actin-cytoskeleton rearrangement. Immune-signaling molecules such as Toll-like receptors, nucleotode-binding oligomerization domain, myeloid differentiation primary response protein 88, and proinflammatory cytokines such as interferon-gamma and tumor necrosis factor-α, play key roles in the induction of innate immune responses. Modifications in B. mallei lipopolysaccharide, in particular, the lipid A acyl groups, stimulate immune responses via Toll-like receptor4 activation that may contribute to persistent infection. SUMMARY: Mortality is high because of septicemia and immune pathogenesis with B. mallei exposure. An effective innate immune response is critical to controlling the acute phase of the infection. Both vaccination and therapeutic approaches are necessary for complete protection against B. mallei.

Burkholderia mallei CLH001 Attenuated Vaccine Strain Is Immunogenic and Protects against Acute Respiratory Glanders.

Burkholderia mallei is the causative agent of glanders, an incapacitating disease with high mortality rates in respiratory cases. Its endemicity and ineffective treatment options emphasize its public health threat and highlight the need for a vaccine. Live attenuated vaccines are considered the most viable vaccine strategy for Burkholderia, but single-gene-deletion mutants have not provided complete protection. In this study, we constructed the select-agent-excluded B. mallei ΔtonB Δhcp1 (CLH001) vaccine strain and investigated its ability to protect against acute respiratory glanders. Here we show that CLH001 is attenuated, safe, and effective at protecting against lethal B. mallei challenge. Intranasal administration of CLH001 to BALB/c and NOD SCID gamma (NSG) mice resulted in complete survival without detectable colonization or abnormal organ histopathology. Additionally, BALB/c mice intranasally immunized with CLH001 in a prime/boost regimen were fully protected against lethal challenge with the B. mallei lux (CSM001) wild-type strain.

Developing Peptide Mimotopes of Capsular Polysaccharides and Lipopolysaccharides Protective Antigens of Pathogenic Burkholderia Bacteria.

Burkholderia pseudomallei (BP) and Burkholderia mallei (BM) are two species of pathogenic Burkholderia bacteria. Our laboratory previously identified four monoclonal antibodies (MAbs) that reacted against Burkholderia capsular polysaccharides (PS) and lipopolysaccharides (LPS) and effectively protected against a lethal dose of BP/BM infections in mice. In this study, we used phage display panning against three different phage peptide libraries to select phage clones specifically recognized by each of the four protective MAbs. After sequencing a total of 179 candidate phage clones, we examined in detail six selected phage clones carrying different peptide inserts for the specificity of binding by the respective target MAbs. Chemically synthesized peptides corresponding to those displayed by the six phage clones were conjugated to keyhole limpet hemocyanin carrier protein and tested for their binding specificity to the respective protective MAbs. The study revealed that four of the six peptides, all derived from the library displaying dodecapeptides, functioned well as "mimotopes" of Burkholderia PS and LPS as demonstrated by a high degree of specific competition against the binding of three protective MAbs to BP and BM. Our results suggest that the four selected peptide mimics corresponding to PS/LPS protective antigens of BP and BM could potentially be developed into peptide vaccines against pathogenic Burkholderia bacteria.

Interlaboratory ring trial to evaluate CFT proficiency of European laboratories for diagnosis of glanders in equids.

To evaluate the routine complement fixation test (CFT) used to detect Burkholderia mallei antibodies in equine sera, an interlaboratory proficiency test was held with 24 European laboratories, including 22 National Reference Laboratories for glanders. The panels sent to participants were composed of sera with or without B mallei antibodies. This study confirmed the reliability of CFT and highlighted its intralaboratory reproducibility. However, the sensitivity of glanders serodiagnosis and laboratory proficiency may be improved by standardising critical reagents, including antigens, and by developing a standard B mallei serum.

Melioidosis and glanders modulation of the innate immune system: barriers to current and future vaccine approaches.

Burkholderia pseudomallei and Burkholderia mallei are pathogenic bacteria causing fatal infections in animals and humans. Both organisms are classified as Tier 1 Select Agents owing to their highly fatal nature, potential/prior use as bioweapons, severity of disease via respiratory exposure, intrinsic resistance to antibiotics, and lack of a current vaccine. Disease manifestations range from acute septicemia to chronic infection, wherein the facultative intracellular lifestyle of these organisms promotes persistence within a broad range of hosts. This ability to thrive intracellularly is thought to be related to exploitation of host immune response signaling pathways. There are currently considerable gaps in our understanding of the molecular strategies employed by these pathogens to modulate these pathways and evade intracellular killing. A better understanding of the specific molecular basis for dysregulation of host immune responses by these organisms will provide a stronger platform to identify novel vaccine targets and develop effective countermeasures.

Phenotypic Characterization of a Novel Virulence-Factor Deletion Strain of Burkholderia mallei That Provides Partial Protection against Inhalational Glanders in Mice.

Burkholderia mallei (Bm) is a highly infectious intracellular pathogen classified as a category B biological agent by the Centers for Disease Control and Prevention. After respiratory exposure, Bm establishes itself within host macrophages before spreading into major organ systems, which can lead to chronic infection, sepsis, and death. Previously, we combined computational prediction of host-pathogen interactions with yeast two-hybrid experiments and identified novel virulence factor genes in Bm, including BMAA0553, BMAA0728 (tssN), and BMAA1865. In the present study, we used recombinant allelic exchange to construct deletion mutants of BMAA0553 and tssN (ΔBMAA0553 and ΔTssN, respectively) and showed that both deletions completely abrogated virulence at doses of >100 times the LD50 of the wild-type Bm strain. Analysis of ΔBMAA0553- and ΔTssN-infected mice showed starkly reduced bacterial dissemination relative to wild-type Bm, and subsequent in vitro experiments characterized pathogenic phenotypes with respect to intracellular growth, macrophage uptake and phagosomal escape, actin-based motility, and multinucleated giant cell formation. Based on observed in vitro and in vivo phenotypes, we explored the use of ΔTssN as a candidate live-attenuated vaccine. Mice immunized with aerosolized ΔTssN showed a 21-day survival rate of 67% after a high-dose aerosol challenge with the wild-type Bm ATCC 23344 strain, compared to a 0% survival rate for unvaccinated mice. However, analysis of histopathology and bacterial burden showed that while the surviving vaccinated mice were protected from acute infection, Bm was still able to establish a chronic infection. Vaccinated mice showed a modest IgG response, suggesting a limited potential of ΔTssN as a vaccine candidate, but also showed prolonged elevation of pro-inflammatory cytokines, underscoring the role of cellular and innate immunity in mitigating acute infection in inhalational glanders.

Characterization of the murine macrophage response to infection with virulent and avirulent Burkholderia species.

BACKGROUND: Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm) are Gram-negative facultative intracellular pathogens, which are the causative agents of melioidosis and glanders, respectively. Depending on the route of exposure, aerosol or transcutaneous, infection by Bp or Bm can result in an extensive range of disease - from acute to chronic, relapsing illness to fatal septicemia. Both diseases are associated with difficult diagnosis and high fatality rates. About ninety five percent of patients succumb to untreated septicemic infections and the fatality rate is 50 % even when standard antibiotic treatments are administered. RESULTS: The goal of this study is to profile murine macrophage-mediated phenotypic and molecular responses that are characteristic to a collection of Bp, Bm, Burkholderia thailandensis (Bt) and Burkholderia oklahomensis (Bo) strains obtained from humans, animals, environment and geographically diverse locations. Burkholderia spp. (N = 21) were able to invade and replicate in macrophages, albeit to varying degrees. All Bp (N = 9) and four Bm strains were able to induce actin polymerization on the bacterial surface following infection. Several Bp and Bm strains showed reduced ability to induce multinucleated giant cell (MNGC) formation, while Bo and Bp 776 were unable to induce this phenotype. Measurement of host cytokine responses revealed a statistically significant Bm mediated IL-6 and IL-10 production compared to Bp strains. Hierarchical clustering of transcriptional data from 84 mouse cytokines, chemokines and their corresponding receptors identified 29 host genes as indicators of differential responses between the Burkholderia spp. Further validation confirmed Bm mediated Il-1b, Il-10, Tnfrsf1b and Il-36a mRNA expressions were significantly higher when compared to Bp and Bt. CONCLUSIONS: These results characterize the phenotypic and immunological differences in the host innate response to pathogenic and avirulent Burkholderia strains and provide insight into the phenotypic alterations and molecular targets underlying host-Burkholderia interactions.

Recombinant Salmonella Expressing Burkholderia mallei LPS O Antigen Provides Protection in a Murine Model of Melioidosis and Glanders.

Burkholderia pseudomallei and Burkholderia mallei are the etiologic agents of melioidosis and glanders, respectively. These bacteria are highly infectious via the respiratory route and can cause severe and often fatal diseases in humans and animals. Both species are considered potential agents of biological warfare; they are classified as category B priority pathogens. Currently there are no human or veterinary vaccines available against these pathogens. Consequently efforts are directed towards the development of an efficacious and safe vaccine. Lipopolysaccharide (LPS) is an immunodominant antigen and potent stimulator of host immune responses. B. mallei express LPS that is structurally similar to that expressed by B. pseudomallei, suggesting the possibility of constructing a single protective vaccine against melioidosis and glanders. Previous studies of others have shown that antibodies against B. mallei or B. pseudomallei LPS partially protect mice against subsequent lethal virulent Burkholderia challenge. In this study, we evaluated the protective efficacy of recombinant Salmonella enterica serovar Typhimurium SL3261 expressing B. mallei O antigen against lethal intranasal infection with Burkholderia thailandensis, a surrogate for biothreat Burkholderia spp. in a murine model that mimics melioidosis and glanders. All vaccine-immunized mice developed a specific antibody response to B. mallei and B. pseudomallei O antigen and to B. thailandensis and were significantly protected against challenge with a lethal dose of B. thailandensis. These results suggest that live-attenuated SL3261 expressing B. mallei O antigen is a promising platform for developing a safe and effective vaccine.