LVS ΔcapB-vectored multiantigenic melioidosis vaccines protect against lethal respiratory Burkholderia pseudomallei challenge in highly sensitive BALB/c mice.

Melioidosis, caused by the intracellular bacterial pathogen and Tier 1 select agent Burkholderia pseudomallei (Bp), is a highly fatal disease endemic in tropical areas. No licensed vaccine against melioidosis exists. In preclinical vaccine studies, demonstrating protection against respiratory infection in the highly sensitive BALB/c mouse has been especially challenging. To address this challenge, we have used a safe yet potent live attenuated platform vector, LVS ΔcapB, previously used successfully to develop vaccines against the Tier 1 select agents of tularemia, anthrax, and plague, to develop a melioidosis vaccine. We have engineered melioidosis vaccines (rLVS ΔcapB/Bp) expressing multiple immunoprotective Bp antigens among type VI secretion system proteins Hcp1, Hcp2, and Hcp6, and membrane protein LolC. Administered intradermally, rLVS ΔcapB/Bp vaccines strongly protect highly sensitive BALB/c mice against lethal respiratory Bp challenge, but protection is overwhelmed at very high challenge doses. In contrast, administered intranasally, rLVS ΔcapB/Bp vaccines remain strongly protective against even very high challenge doses. Under some conditions, the LVS ΔcapB vector itself provides significant protection against Bp challenge, and consistent with this, both the vector and vaccines induce humoral immune responses to Bp antigens. Three-antigen vaccines expressing Hcp6-Hcp1-Hcp2 or Hcp6-Hcp1-LolC are among the most potent and provide long-term protection and protection even with a single intranasal immunization. Protection via the intranasal route was either comparable to or statistically significantly better than the single-deletional Bp mutant Bp82, which served as a positive control. Thus, rLVS ΔcapB/Bp vaccines are exceptionally promising safe and potent melioidosis vaccines. IMPORTANCE: Melioidosis, a major neglected disease caused by the intracellular bacterial pathogen Burkholderia pseudomallei, is endemic in many tropical areas of the world and causes an estimated 165,000 cases and 89,000 deaths in humans annually. Moreover, B. pseudomallei is categorized as a Tier 1 select agent of bioterrorism, largely because inhalation of low doses can cause rapidly fatal pneumonia. No licensed vaccine is available to prevent melioidosis. Here, we describe a safe and potent melioidosis vaccine that protects against lethal respiratory challenge with B. pseudomallei in a highly sensitive small animal model-even a single immunization is highly protective, and the vaccine gives long-term protection. The vaccine utilizes a highly attenuated replicating intracellular bacterium as a vector to express multiple key proteins of B. pseudomallei; this vector platform has previously been used successfully to develop potent vaccines against other Tier 1 select agent diseases including tularemia, anthrax, and plague.

Early innate and adaptive immune perturbations determine long-term severity of chronic virus and Mycobacterium tuberculosis coinfection.

Chronic viral infections increase severity of Mycobacterium tuberculosis (Mtb) coinfection. Here, we examined how chronic viral infections alter the pulmonary microenvironment to foster coinfection and worsen disease severity. We developed a coordinated system of chronic virus and Mtb infection that induced central clinical manifestations of coinfection, including increased Mtb burden, extra-pulmonary dissemination, and heightened mortality. These disease states were not due to chronic virus-induced immunosuppression or exhaustion; rather, increased amounts of the cytokine TNFα initially arrested pulmonary Mtb growth, impeding dendritic cell mediated antigen transportation to the lymph node and subverting immune-surveillance, allowing bacterial sanctuary. The cryptic Mtb replication delayed CD4 T cell priming, redirecting T helper (Th) 1 toward Th17 differentiation and increasing pulmonary neutrophilia, which diminished long-term survival. Temporally restoring CD4 T cell induction overcame these diverse disease sequelae to enhance Mtb control. Thus, Mtb co-opts TNFα from the chronic inflammatory environment to subvert immune-surveillance, avert early immune function, and foster long-term coinfection.

Nanoparticle Formulation of Moxifloxacin and Intramuscular Route of Delivery Improve Antibiotic Pharmacokinetics and Treatment of Pneumonic Tularemia in a Mouse Model.

Francisella tularensis causes a serious and often fatal infection, tularemia. We compared the efficacy of moxifloxacin formulated as free drug vs disulfide snap-top mesoporous silica nanoparticles (MSNs) in a mouse model of pneumonic tularemia. We found that MSN-formulated moxifloxacin was more effective than free drug and that the intramuscular and subcutaneous routes were markedly more effective than the intravenous route. Measurement of tissue silica levels and fluorescent flow cytometry assessment of colocalization of MSNs with infected cells revealed that the enhanced efficacy of MSNs and the intramuscular route of delivery was not due to better delivery of MSNs to infected tissues or cells. However, moxifloxacin blood levels demonstrated that the nanoparticle formulation and intramuscular route provided the longest half-life and longest time above the minimal inhibitory concentration. Thus, improved pharmacokinetics are responsible for the greater efficacy of nanoparticle formulation and intramuscular delivery compared with free drug and intravenous delivery.

PPE37 Is Essential for Mycobacterium tuberculosis Heme-Iron Acquisition (HIA), and a Defective PPE37 in Mycobacterium bovis BCG Prevents HIA.

Mycobacterium tuberculosis, one of the world's leading causes of death, must acquire nutrients, such as iron, from the host to multiply and cause disease. Iron is an essential metal and M. tuberculosis possesses two different systems to acquire iron from its environment: siderophore-mediated iron acquisition (SMIA) and heme-iron acquisition (HIA), involving uptake and degradation of heme to release ferrous iron. We have discovered that Mycobacterium bovis BCG, the tuberculosis vaccine strain, is severely deficient in HIA, and we exploited this phenotypic difference between BCG and M. tuberculosis to identify genes involved in HIA by complementing BCG's defect with a fosmid library. We identified ppe37, an iron-regulated PPE family gene, as being essential for HIA. BCG complemented with M. tuberculosisppe37 exhibits HIA as efficient as that of M. tuberculosis, achieving robust growth with <0.2 µM hemin. Conversely, deletion of ppe37 from M. tuberculosis results in a strain severely attenuated in HIA, with a phenotype nearly identical to that of BCG, requiring a 200-fold higher concentration of hemin to achieve growth equivalent to that of its parental strain. A nine-amino-acid deletion near the N terminus of BCG PPE37 (amino acids 31 to 39 of the M. tuberculosis PPE37 protein) underlies BCG's profound defect in HIA. Significant genetic variability exists in ppe37 genes across different M. tuberculosis strains, with more than 60% of sequences from completely sequenced M. tuberculosis genomes having mutations that result in altered PPE37 proteins; furthermore, these altered PPE37 proteins are nonfunctional in HIA. Our findings should allow delineation of the relative roles of HIA and SMIA in M. tuberculosis pathogenesis.

Type I and Type II Interferon Coordinately Regulate Suppressive Dendritic Cell Fate and Function during Viral Persistence.

Persistent viral infections are simultaneously associated with chronic inflammation and highly potent immunosuppressive programs mediated by IL-10 and PDL1 that attenuate antiviral T cell responses. Inhibiting these suppressive signals enhances T cell function to control persistent infection; yet, the underlying signals and mechanisms that program immunosuppressive cell fates and functions are not well understood. Herein, we use lymphocytic choriomeningitis virus infection (LCMV) to demonstrate that the induction and functional programming of immunosuppressive dendritic cells (DCs) during viral persistence are separable mechanisms programmed by factors primarily considered pro-inflammatory. IFNγ first induces the de novo development of naive monocytes into DCs with immunosuppressive potential. Type I interferon (IFN-I) then directly targets these newly generated DCs to program their potent T cell immunosuppressive functions while simultaneously inhibiting conventional DCs with T cell stimulating capacity. These mechanisms of monocyte conversion are constant throughout persistent infection, establishing a system to continuously interpret and shape the immunologic environment. MyD88 signaling was required for the differentiation of suppressive DCs, whereas inhibition of stimulatory DCs was dependent on MAVS signaling, demonstrating a bifurcation in the pathogen recognition pathways that promote distinct elements of IFN-I mediated immunosuppression. Further, a similar suppressive DC origin and differentiation was also observed in Mycobacterium tuberculosis infection, HIV infection and cancer. Ultimately, targeting the underlying mechanisms that induce immunosuppression could simultaneously prevent multiple suppressive signals to further restore T cell function and control persistent infections.

rBCG30-induced immunity and cross-protection against Mycobacterium leprae challenge are enhanced by boosting with the Mycobacterium tuberculosis 30-kilodalton antigen 85B.

Leprosy remains a major global health problem and typically occurs in regions in which tuberculosis is endemic. Vaccines are needed that protect against both infections and do so better than the suboptimal Mycobacterium bovis BCG vaccine. Here, we evaluated rBCG30, a vaccine previously demonstrated to induce protection superior to that of BCG against Mycobacterium tuberculosis and Mycobacterium bovis challenge in animal models, for efficacy against Mycobacterium leprae challenge in a murine model of leprosy. rBCG30 overexpresses the M. tuberculosis 30-kDa major secretory protein antigen 85B, which is 85% homologous with the M. leprae homolog (r30ML). Mice were sham immunized or immunized intradermally with BCG or rBCG30 and challenged 2.5 months later by injection of viable M. leprae into each hind footpad. After 7 months, vaccine efficacy was assessed by enumerating the M. leprae bacteria per footpad. Both BCG and rBCG30 induced significant protection against M. leprae challenge. In the one experiment in which a comparison between BCG and rBCG30 was feasible, rBCG30 induced significantly greater protection than did BCG. Immunization of mice with purified M. tuberculosis or M. leprae antigen 85B also induced protection against M. leprae challenge but less so than BCG or rBCG30. Notably, boosting rBCG30 with M. tuberculosis antigen 85B significantly enhanced r30ML-specific immune responses, substantially more so than boosting BCG, and significantly augmented protection against M. leprae challenge. Thus, rBCG30, a vaccine that induces improved protection against M. tuberculosis, induces cross-protection against M. leprae that is comparable or potentially superior to that induced by BCG, and boosting rBCG30 with antigen 85B further enhances immune responses and protective efficacy.

Characterization of a Mycobacterium tuberculosis nanocompartment and its potential cargo proteins.

Mycobacterium tuberculosis has evolved various mechanisms by which the bacterium can maintain homeostasis under numerous environmental assaults generated by the host immune response. M. tuberculosis harbors enzymes involved in the oxidative stress response that aid in survival during the production of reactive oxygen species in activated macrophages. Previous studies have shown that a dye-decolorizing peroxidase (DyP) is encapsulated by a bacterial nanocompartment, encapsulin (Enc), whereby packaged DyP interacts with Enc via a unique C-terminal extension. M. tuberculosis also harbors an encapsulin homolog (CFP-29, Mt-Enc), within an operon with M. tuberculosis DyP (Mt-DyP), which contains a C-terminal extension. Together these observations suggest that Mt-DyP interacts with Mt-Enc. Furthermore, it has been suggested that DyPs may function as either a heme-dependent peroxidase or a deferrochelatase. Like Mt-DyP, M. tuberculosis iron storage ferritin protein, Mt-BfrB, and an M. tuberculosis protein involved in folate biosynthesis, 7,8-dihydroneopterin aldolase (Mt-FolB), have C-terminal tails that could also interact with Mt-Enc. For the first time, we show by co-purification and electron microscopy that mycobacteria via Mt-Enc can encapsulate Mt-DyP, Mt-BfrB, and Mt-FolB. Functional studies of free or encapsulated proteins demonstrate that they retain their enzymatic activity within the Mt-Enc nanocompartment. Mt-DyP, Mt-FolB, and Mt-BfrB all have antioxidant properties, suggesting that if these proteins are encapsulated by Mt-Enc, then this nanocage may play a role in the M. tuberculosis oxidative stress response. This report provides initial structural and biochemical clues regarding the molecular mechanisms that utilize compartmentalization by which the mycobacterial cell may aid in detoxification of the local environment to ensure long term survival.

Discovery and characterization of a unique mycobacterial heme acquisition system.

Mycobacterium tuberculosis must import iron from its host for survival, and its siderophore-dependent iron acquisition pathways are well established. Here we demonstrate a newly characterized pathway, whereby M. tuberculosis can use free heme and heme from hemoglobin as an iron source. Significantly, we identified the genomic region, Rv0202c-Rv0207c, responsible for the passage of heme iron across the mycobacterial membrane. Key players of this heme uptake system were characterized including a secreted protein and two transmembrane proteins, all three specific to mycobacteria. Furthermore, the crystal structure of the key heme carrier protein Rv0203 was found to have a unique fold. The discovery of a unique mycobacterial heme acquisition pathway opens new avenues of exploration into mycobacterial therapeutics.

A Replication-Limited Recombinant Mycobacterium bovis BCG vaccine against tuberculosis designed for human immunodeficiency virus-positive persons is safer and more efficacious than BCG.

Tuberculosis is the leading cause of death in AIDS patients, yet the current tuberculosis vaccine, Mycobacterium bovis bacillus Calmette-Guérin (BCG), is contraindicated for immunocompromised individuals, including human immunodeficiency virus-positive persons, because it can cause disseminated disease; moreover, its efficacy is suboptimal. To address these problems, we have engineered BCG mutants that grow normally in vitro in the presence of a supplement, are preloadable with supplement to allow limited growth in vivo, and express the highly immunoprotective Mycobacterium tuberculosis 30-kDa major secretory protein. The limited replication in vivo renders these vaccines safer than BCG in SCID mice yet is sufficient to induce potent cell-mediated and protective immunity in the outbred guinea pig model of pulmonary tuberculosis. In the case of one vaccine, rBCG(mbtB)30, protection was superior to that with BCG (0.3-log fewer CFU of M. tuberculosis in the lung [P < 0.04] and 0.6-log fewer CFU in the spleen [P = 0.001] in aerosol-challenged animals [means for three experiments]); hence, rBCG(mbtB)30 is the first live mycobacterial vaccine that is both more attenuated than BCG in the SCID mouse and more potent than BCG in the guinea pig. Our study demonstrates the feasibility of developing safer and more potent vaccines against tuberculosis. The novel approach of engineering a replication-limited vaccine expressing a recombinant immunoprotective antigen and preloading it with a required nutrient, such as iron, that is capable of being stored should be generally applicable to other live vaccine vectors targeting intracellular pathogens.

All four Mycobacterium tuberculosis glnA genes encode glutamine synthetase activities but only GlnA1 is abundantly expressed and essential for bacterial homeostasis.

Glutamine synthetases (GS) are ubiquitous enzymes that play a central role in every cell's nitrogen metabolism. We have investigated the expression and activity of all four genomic Mycobacterium tuberculosis GS - GlnA1, GlnA2, GlnA3 and GlnA4 - and four enzymes regulating GS activity and/or nitrogen and glutamate metabolism - adenylyl transferase (GlnE), gamma-glutamylcysteine synthase (GshA), UDP-N-acetylmuramoylalanine-D-glutamate ligase (MurD) and glutamate racemase (MurI). All eight genes are located in multigene operons except for glnA1, and all are transcribed in M. tuberculosis; however, some are not translated or translated at such low levels that the enzymes escape detection. Of the four GS, only GlnA1 can be detected. Each of the eight genes, as well as the glnA1-glnE-glnA2 cluster, was expressed separately in Mycobacterium smegmatis, and its gene product was characterized and assayed for enzymatic activity by analysing the reaction products. In M. smegmatis, all four recombinant-overexpressed GS are multimeric enzymes exhibiting GS activity. Whereas GlnA1, GlnA3 and GlnA4 catalyse the synthesis of L-glutamine, GlnA2 catalyses the synthesis of D-glutamine and D-isoglutamine. The generation of mutants in M. tuberculosis of the four glnA genes, murD and murI demonstrated that all of these genes except glnA1 are nonessential for in vitro growth. L-methionine-S,R-sulphoximine (MSO), previously demonstrated to inhibit M. tuberculosis growth in vitro and in vivo, strongly inhibited all four GS enzymes; hence, the design of MSO analogues with an improved therapeutic to toxic ratio remains a promising strategy for the development of novel anti-M. tuberculosis drugs.

Glutamine synthetase GlnA1 is essential for growth of Mycobacterium tuberculosis in human THP-1 macrophages and guinea pigs.

To assess the role of glutamine synthetase (GS), an enzyme of central importance in nitrogen metabolism, in the pathogenicity of Mycobacterium tuberculosis, we constructed a glnA1 mutant via allelic exchange. The mutant had no detectable GS protein or GS activity and was auxotrophic for L-glutamine. In addition, the mutant was attenuated for intracellular growth in human THP-1 macrophages and avirulent in the highly susceptible guinea pig model of pulmonary tuberculosis. Based on growth rates of the mutant in the presence of various concentrations of L-glutamine, the effective concentration of L-glutamine in the M. tuberculosis phagosome of THP-1 cells was approximately 10% of the level assayed in the cytoplasm of these cells (4.5 mM), indicating that the M. tuberculosis phagosome is impermeable to even very small molecules in the macrophage cytoplasm. When complemented by the M. tuberculosis glnA1 gene, the mutant exhibited a wild-type phenotype in broth culture and in human macrophages, and it was virulent in guinea pigs. When complemented by the Salmonella enterica serovar Typhimurium glnA gene, the mutant had only 1% of the GS activity of the M. tuberculosis wild-type strain because of poor expression of the S. enterica serovar Typhimurium GS in the heterologous M. tuberculosis host. Nevertheless, the strain complemented with S. enterica serovar Typhimurium GS grew as well as the wild-type strain in broth culture and in human macrophages. This strain was virulent in guinea pigs, although somewhat less so than the wild-type. These studies demonstrate that glnA1 is essential for M. tuberculosis virulence.

Metabolic incorporation of unnatural sialic acids into Haemophilus ducreyi lipooligosaccharides.

The lipooligosaccharides (LOS) of Haemophilus ducreyi are highly sialylated, a modification that has been implicated in resistance to host defense and in virulence. In previous work, we demonstrated that H. ducreyi scavenges sialic acid from the extracellular milieu and incorporates those residues into LOS. Here we report that H. ducreyi can use unnatural sialic acids bearing elongated N-acyl groups from three to seven carbon atoms in length, resulting in outer membrane presentation of unnatural sialyl-LOS. The unnatural variant comprises approximately 90% of cell surface sialosides when exogenous substrates are added to the media at micromolar concentrations, despite the availability of natural sialic acid in the growth media. Although they represent the majority of cell surface sialosides, analogs with longer N-acyl groups diminish the overall level of LOS sialylation, culminating in complete inhibition of LOS sialylation by N-octanoyl sialic acid. Thus, sialylation of H. ducreyi LOS can be modulated with respect to the structure of the terminal sialic acid residue and the extent to which the LOS acceptor is modified by supplying the bacteria with various sialic acid analogs.

The lbgAB gene cluster of Haemophilus ducreyi encodes a beta-1,4-galactosyltransferase and an alpha-1,6-DD-heptosyltransferase involved in lipooligosaccharide biosynthesis.

All Haemophilus ducreyi strains examined contain a lipooligosaccharide (LOS) consisting of a single but variable branch oligosaccharide that emanates off the first heptose (Hep-I) of a conserved Hep(3)-phosphorylated 3-deoxy-D-manno-octulosonic acid-lipid A core. In a previous report, identification of tandem genes, lbgA and lbgB, that are involved in LOS biosynthesis was described (Stevens et al., Infect. Immun. 65:651-660, 1997). In a separate study, the same gene cluster was identified and the lbgB (losB) gene was found to be required for transfer of the second sugar, D-glycero-D-manno-heptose (DD-Hep), of the major branch structure (Gibson et al., J. Bacteriol. 179:5062-5071, 1997). In this study, we identified the function of the neighboring upstream gene, lbgA, and found that it is necessary for addition of the third sugar in the dominant oligosaccharide branch, a galactose-linked beta1-->4, to the DD-Hep. LOS from an lbgA mutant and an lbgAB double mutant were isolated and were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, carbohydrate analysis, mass spectrometry, and nuclear magnetic resonance spectroscopy. The results showed that the mutant strains synthesize truncated LOS glycoforms that terminate after addition of the first glucose (lbgAB) or the disaccharide DD-Hepalpha1-->6Glcbeta1 (lbgA) that is attached to the heptose core. Both mutants show a significant reduction in the ability to adhere to human keratinocytes. Although minor differences were observed after two-dimensional gel electrophoresis of total proteins from the wild-type and mutant strains, the expression levels of the vast majority of proteins were unchanged, suggesting that the differences in adherence and invasion are due to differences in LOS. These studies add to the mounting evidence for a role of full-length LOS structures in the pathophysiology of H. ducreyi infection.