Development of vaccines against burkholderia pseudomallei.

Burkholderia pseudomallei is a Gram-negative bacterium which is the causative agent of melioidosis, a disease which carries a high mortality and morbidity rate in endemic areas of South East Asia and Northern Australia. At present there is no available human vaccine that protects against B. pseudomallei, and with the current limitations of antibiotic treatment, the development of new preventative and therapeutic interventions is crucial. This review considers the multiple elements of melioidosis vaccine research including: (i) the immune responses required for protective immunity, (ii) animal models available for preclinical testing of potential candidates, (iii) the different experimental vaccine strategies which are being pursued, and (iv) the obstacles and opportunities for eventual registration of a licensed vaccine in humans.

Combining vaccination and postexposure CpG therapy provides optimal protection against lethal sepsis in a biodefense model of human melioidosis.

The Gram-negative bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a major cause of lethal sepsis and morbidity in endemic areas of Southeast Asia and a potential bioterrorism threat. We have used susceptible BALB/c mice to evaluate the potential of targeting vaccination and generic immunotherapy to the lung for optimal protection against respiratory challenge. Intranasal vaccination with live attenuated B. pseudomallei increased survival and induced interferon-γ-secreting T cells in the lung. Intranasal delivery of CpG oligodeoxynucleotides also provided significant protection; however, combining preexposure vaccination with CpG treatment at the time of infection or up to 18 hours after infection, provided significantly greater protection than either treatment alone. This combination prolonged survival, decreased bacterial loads by >1000-fold, and delayed the onset of sepsis. This novel approach may be applicable to other potential biodefense agents for which existing countermeasures are not fully effective.

Burkholderia pseudomallei: animal models of infection.

A range of animal models of Burkholderia pseudomallei infection have been reported, and the host species differ widely both in their susceptibility to infection and in the pathogenesis of disease. In mice, and depending on the route of infection, dose, and mouse strain, the disease can range from a chronic, and in some cases, an apparently latent infection to an acute fulminant disease. Alternative small animal models of infection include diabetic rats or hamsters. Larger animal models of disease have not yet been fully developed. It is not clear which of the small animal models of melioidosis most accurately reflect disease in humans. However, the findings that diabetic rats are susceptible to infection, that some strains of mice can develop persistent subclinical infections that can spontaneously reactivate, and that inhalation exposure generally results in more acute disease suggest that these different models mimic different aspects of human melioidosis.

Altered proteome in Burkholderia pseudomallei rpoE operon knockout mutant: insights into mechanisms of rpoE operon in stress tolerance, survival, and virulence.

We have previously shown that the alternative sigma factor sigmaE (RpoE), encoded by rpoE, is involved in stress tolerance and survival of Burkholderia pseudomallei. However, its molecular and pathogenic mechanisms remain unclear. In the present study, we applied gel-based, differential proteomics to compare the cellular proteome of an rpoE operon knockout mutant (RpoE Mut) to that of wild-type (K96243 WT) B. pseudomallei. Quantitative intensity analysis (n = 5 gels from 5 individual culture flasks in each group) revealed significantly differential expression of 52 proteins, which were subsequently identified by Q-TOF MS/MS. These included oxidative, osmotic, and other stress response proteins; chaperones; transcriptional/translational regulators; metabolic enzymes; proteins involved in cell wall synthesis, fatty synthesis, glycogen synthesis, and storage; exported proteins; secreted proteins; adhesion molecule; protease/peptidase; protease inhibitor; signaling proteins; and other miscellaneous proteins. The down-regulation of several stress response proteins, chaperones, transcriptional/translational regulators, and proteins involved in cell wall synthesis in RpoE Mut provided some new insights into the mechanisms of the rpoE operon for the stress tolerance and survival of B. pseudomallei. In addition, the proteomic data and in vivo study indicated that the rpoE operon is also involved in the virulence of B. pseudomallei. Our findings underscore the usefulness of proteomics for unraveling pathogenic mechanisms of diseases at the molecular level.

A critical role for neutrophils in resistance to experimental infection with Burkholderia pseudomallei.

Inhalation is an important route of infection with Burkholderia pseudomallei, the causative agent of melioidosis. In resistant C57BL/6 mice, activated neutrophils are rapidly recruited to the lungs after intranasal B. pseudomallei infection. Prevention of this response by use of the anti-Gr-1+ cell-depleting monoclonal antibody RB6-8C5 severely exacerbated disease, resulting in an acute lethal infection associated with a 1000-fold increase in lung bacterial loads within 4 days. C57BL/6 interferon (IFN)-gamma(-/-) mice were also acutely susceptible to pulmonary B. pseudomallei infection, dying within 3 days of challenge; this suggests that IFN-gamma is essential for control in the lungs and precedes the protective role of neutrophils in resistance. In neutrophil-depleted mice, lung concentrations of tumor necrosis factor (TNF)-alpha, IFN-gamma, and interleukin-6 were decreased by up to 98%. Natural killer cells were the principle source of IFN-gamma, and monocytes were the principle source of TNF-alpha, suggesting that neutrophils play an important indirect role in the generation of the early cytokine environment in the lungs.

A live experimental vaccine against Burkholderia pseudomallei elicits CD4+ T cell-mediated immunity, priming T cells specific for 2 type III secretion system proteins.

Burkholderia pseudomallei is the etiological agent of melioidosis, a serious human disease for which no vaccine is available. Immunization of susceptible BALB/c mice with the live attenuated mutant B. pseudomallei ilvI (referred to as "2D2") generated significant, although incomplete, immunity. Splenic B. pseudomallei-specific T cells, detected in immunized mice, proliferated and produced interferon-gamma in vitro in response to dead bacteria. Assessment of T cell antigen specificity indicated that subpopulations of B. pseudomallei-reactive T cells were responsive to BopE, a type III secretion system (TTSS) effector protein, and to a lesser extent to BipD, a TTSS translocator protein. Increased survival of severe combined immunodeficient mice adoptively transferred with T cells from immunized mice, compared with that of naive T cell recipients, demonstrated that immunization with 2D2 generated T cell-mediated immunity. CD4+ and CD8+ cell depletion studies demonstrated that CD4+ cells, but not CD8+ cells, mediated this protection in vivo. Thus, CD4+ T cells can mediate vaccine-induced immunity to experimental melioidosis.

Role of T cells in innate and adaptive immunity against murine Burkholderia pseudomallei infection.

Antigen-specific T cells are important sources of interferon (IFN)-gamma for acquired immunity to intracellular pathogens, but they can also produce IFN- gamma directly via a "bystander" activation pathway in response to proinflammatory cytokines. We investigated the in vivo role of cytokine- versus antigen-mediated T cell activation in resistance to the pathogenic bacterium Burkholderia pseudomallei. IFN-gamma, interleukin (IL)-12, and IL-18 were essential for initial bacterial control in infected mice. B. pseudomallei infection rapidly generated a potent IFN-gamma response from natural killer (NK) cells, NK T cells, conventional T cells, and other cell types within 16 h after infection, in an IL-12- and IL-18-dependent manner. However, early T cell- and NK cell-derived IFN-gamma responses were functionally redundant in cell depletion studies, with IFN-gamma produced by other cell types, such as major histocompatibility complex class II(int) F4/80(+) macrophages being sufficient for initial resistance. In contrast, B. pseudomallei-specific CD4(+) T cells played an important role during the later stage of infection. Thus, the T cell response to primary B. pseudomallei infection is biphasic, an early cytokine-induced phase in which T cells appear to be functionally redundant for initial bacterial clearance, followed by a later antigen-induced phase in which B. pseudomallei-specific T cells, in particular CD4(+) T cells, are important for host resistance.

Plasmodium falciparum: stage specific effects of a selective inhibitor of lactate dehydrogenase.

Plasmodium falciparum lactate dehydrogenase (PfLDH) is essential for ATP generation. Based on structural differences within the active site between P. falciparum and human LDH, we have identified a series of heterocyclic azole-based inhibitors that selectively bind within the PfLDH but not the human LDH (hLDH) active site and showed anti-malarial activity in vitro and in vivo. Here we expand on an azole, OXD1, from this series and found that the anti-P. falciparum activity was retained against a panel of strains independently of their anti-malarial drug sensitivity profile. Trophozoites had relatively higher PfLDH enzyme activity and PfLDH-RNA expression levels than rings and were the most susceptible stages to OXD1 exposure. This is probably linked to their increased energy requirements and consistent with glycolysis being an essential metabolic pathway for parasite survival within the erythrocyte. Further structural elaboration of these azoles could lead to the identification of compounds that target P. falciparum through such a novel mechanism and with more potent anti-malarial activity.

Mapping the binding site for gossypol-like inhibitors of Plasmodium falciparum lactate dehydrogenase.

Gossypol is a di-sesquiterpene natural-product in the form of a functionalised binaphthyl and is isolated from cotton plants. The compound has long been known to exhibit anti-malarial and other biological activities. Previous studies have indicated that compounds of this type target Plasmodium falciparum lactate dehydrogenase (pfLDH), an essential enzyme for energy generation within the parasite. In this study, we report that simple naphthalene-based compounds, the core of the gossypol structure, exhibit weak inhibition of the parasite lactate dehydrogenase. Crystal structures of the complexes formed by binding of these naphthalene-based compounds to their target enzyme have been used to delineate the molecular features likely to form the gossypol binding site. Two modes of binding are observed: one overlapping the pyruvate but not the co-factor site, the other bridging the binding sites for the co-factor nicontinamide group and pyruvate substrate. This latter site encompasses molecular features unique to Plasmodium forms of LDH and is likely to represent the mode of binding for gossypol derivatives that show selectivity for the parasite enzymes. We also report a substrate analogue that unexpectedly binds within the adenine pocket of the co-factor groove. Although these core pharmacophore-like molecules only exhibit low levels of inhibitory activity, these molecular snapshots provide a rational basis for renewed structure-based development of naphthalene-based compounds as anti-malarial agents.

Attenuated virulence and protective efficacy of a Burkholderia pseudomallei bsa type III secretion mutant in murine models of melioidosis.

Melioidosis is a severe infectious disease of animals and humans caused by the Gram-negative intracellular pathogen Burkholderia pseudomallei. An Inv/Mxi-Spa-like type III protein secretion apparatus, encoded by the B. pseudomallei bsa locus, facilitates bacterial invasion of epithelial cells, escape from endocytic vesicles and intracellular survival. This study investigated the role of the Bsa type III secretion system in the pathogenesis of melioidosis in murine models. B. pseudomallei bipD mutants, lacking a component of the translocation apparatus, were found to be significantly attenuated following intraperitoneal or intranasal challenge of BALB/c mice. Furthermore, a bipD mutant was attenuated in C57BL/6 IL-12 p40(-/-) mice, which are highly susceptible to B. pseudomallei infection. Mutation of bipD impaired bacterial replication in the liver and spleen of BALB/c mice in the early stages of infection. B. pseudomallei mutants lacking either the type III secreted guanine nucleotide exchange factor BopE or the putative effectors BopA or BopB exhibited varying degrees of attenuation, with mutations in bopA and bopB causing a significant delay in median time to death. This indicates that bsa-encoded type III secreted proteins may act in concert to determine the outcome of B. pseudomallei infection in mice. Mice inoculated with the B. pseudomallei bipD mutant were partially protected against subsequent challenge with wild-type B. pseudomallei. However, immunization of mice with purified BipD protein was not protective.

Identification and activity of a series of azole-based compounds with lactate dehydrogenase-directed anti-malarial activity.

Plasmodium falciparum, the causative agent of malaria, relies extensively on glycolysis coupled with homolactic fermentation during its blood-borne stages for energy production. Selective inhibitors of the parasite lactate dehydrogenase (LDH), central to NAD(+) regeneration, therefore potentially provide a route to new antimalarial drugs directed against a novel molecular target. A series of heterocyclic, azole-based compounds are described that preferentially inhibit P. falciparum LDH at sub-micromolar concentrations, typically at concentrations about 100-fold lower than required for human lactate dehydrogenase inhibition. Crystal structures show these competitive inhibitors form a network of interactions with amino acids within the active site of the enzyme, stacking alongside the nicotinamide ring of the NAD(+) cofactor. These compounds display modest activity against parasitized erythrocytes, including parasite strains with known resistance to existing anti-malarials and against Plasmodium berghei in BALB/c mice. Initial toxicity data suggest the azole derivatives have generally low cytotoxicity, and preliminary pharmoco-kinetic data show favorable bioavailability and circulation times. These encouraging results suggest that further enhancement of these structures may yield candidates suitable for consideration as new therapeutics for the treatment of malaria. In combination these studies also provide strong support for the validity of targeting the Plasmodium glycolytic pathway and, in particular, LDH in the search for novel anti-malarials.