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.

The rLVS ΔcapB/iglABC vaccine provides potent protection in Fischer rats against inhalational tularemia caused by various virulent Francisella tularensis strains.

Francisella tularensis is one of the several biothreat agents for which a licensed vaccine is needed. To ensure vaccine protection is achieved across a range of virulent F. tularensis strains, we assembled and characterized a panel of F. tularensis isolates to be utilized as challenge strains. A promising tularemia vaccine candidate is rLVS ΔcapB/iglABC (rLVS), in which the vector is the LVS strain with a deletion in the capB gene and which additionally expresses a fusion protein comprising immunodominant epitopes of proteins IglA, IglB, and IglC. Fischer rats were immunized subcutaneously 1-3 times at 3-week intervals with rLVS at various doses. The rats were exposed to a high dose of aerosolized Type A strain Schu S4 (FRAN244), a Type B strain (FRAN255), or a tick derived Type A strain (FRAN254) and monitored for survival. All rLVS vaccination regimens including a single dose of 107 CFU rLVS provided 100% protection against both Type A strains. Against the Type B strain, two doses of 107 CFU rLVS provided 100% protection, and a single dose of 107 CFU provided 87.5% protection. In contrast, all unvaccinated rats succumbed to aerosol challenge with all of the F. tularensis strains. A robust Th1-biased antibody response was induced in all vaccinated rats against all F. tularensis strains. These results demonstrate that rLVS ΔcapB/iglABC provides potent protection against inhalational challenge with either Type A or Type B F. tularensis strains and should be considered for further analysis as a future tularemia vaccine.

Efficacy and safety of an innovative short-course regimen containing clofazimine for treatment of drug-susceptible tuberculosis: a clinical trial.

In preclinical studies, a new antituberculosis drug regimen markedly reduced the time required to achieve relapse-free cure. This study aimed to preliminarily evaluate the efficacy and safety of this four-month regimen, consisting of clofazimine, prothionamide, pyrazinamide and ethambutol, with a standard six-month regimen in patients with drug-susceptible tuberculosis. An open-label pilot randomized clinical trial was conducted among the patients with newly diagnosed bacteriologically-confirmed pulmonary tuberculosis. The primary efficacy end-point was sputum culture negative conversion. Totally, 93 patients were included in the modified intention-to-treat population. The rates of sputum culture conversion were 65.2% (30/46) and 87.2% (41/47) for short-course and standard regimen group, respectively. There was no difference on two-month culture conversion rates, time to culture conversion, nor early bactericidal activity (P > 0.05). However, patients on short-course regimen were observed with lower rates of radiological improvement or recovery and sustained treatment success, which was mainly attributed to higher percent of patients permanently changed assigned regimen (32.1% vs. 12.3%, P = 0.012). The main cause for it was drug-induced hepatitis (16/17). Although lowering the dose of prothionamide was approved, the alternative option of changing assigned regimen was chosen in this study. While in per-protocol population, sputum culture conversion rates were 87.0% (20/23) and 94.4% (34/36) for the respective groups. Overall, the short-course regimen appeared to have inferior efficacy and higher incidence of hepatitis but desired efficacy in per-protocol population. It provides the first proof-of-concept in humans of the capacity of the short-course approach to identify drug regimens that can shorten the treatment time for tuberculosis.

Oral Administration of Universal Bacterium-Vectored Nucleocapsid-Expressing COVID-19 Vaccine is Efficacious in Hamsters.

Oral delivery of an inexpensive COVID-19 (coronavirus disease 2019) vaccine could dramatically improve immunization rates, especially in low- and middle-income countries. Previously, we described a potential universal COVID-19 vaccine, rLVS ΔcapB/MN, comprising a replicating bacterial vector, LVS (live vaccine strain) ΔcapB, expressing the highly conserved SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) membrane and nucleocapsid (N) proteins, which, when administered intradermally or intranasally, protects hamsters from severe COVID-19-like disease after high-dose SARS-CoV-2 respiratory challenge. Here, we show that oral administration of the vaccine also protects against high-dose SARS-CoV-2 respiratory challenge; its protection is comparable to that of intradermal, intranasal, or subcutaneous administration. Hamsters were protected against severe weight loss and lung pathology and had reduced oropharyngeal and lung virus titers. Protection against weight loss and histopathology by the vaccine, which in mice induces splenic and lung cell interferon gamma in response to N protein stimulation, was correlated in hamsters with pre-challenge serum anti-N TH1-biased IgG (IgG2/3). Thus, rLVS ΔcapB/MN has potential as an oral universal COVID-19 vaccine. IMPORTANCE The COVID-19 pandemic continues to rage into its fourth year worldwide. To protect the world's population most effectively from severe disease, hospitalization, and death, a vaccine is needed that is resistant to rapidly emerging viral variants of the causative agent SARS-CoV-2, inexpensive to manufacture, store, and transport, and easy to administer. Ideally, such a vaccine would be capable of oral administration, especially in resource-poor countries of the world where there are shortages of needles, syringes and trained personnel to administer injectable vaccines. Here, we show that oral administration of a bacterium-vectored vaccine meeting all these criteria protects naturally susceptible Syrian hamsters from severe COVID-19-like disease, including severe weight loss and lung pathology, after high-dose SARS-CoV-2 respiratory challenge. As the vaccine is based upon inducing immunity to highly conserved SARS-CoV-2 membrane and nucleocapsid proteins, as opposed to the rapidly mutating Spike protein, it should remain resistant to newly emerging SARS-CoV-2 variants.

Listeria-vectored multi-antigenic tuberculosis vaccine protects C57BL/6 and BALB/c mice and guinea pigs against Mycobacterium tuberculosis challenge.

Mycobacterium tuberculosis (Mtb) infects one-third of the world's population and is a leading cause of death from a single infectious agent. New TB vaccines are urgently needed to augment immunity conferred by the current modestly protective BCG vaccine. We have developed live attenuated recombinant Listeria monocytogenes (rLm)-vectored TB vaccines expressing five [Mpt64/23.5-EsxH/TB10.4-EsxA/ESAT6-EsxB/CFP10-Ag85B/r30] (rLmMtb5Ag) or nine (additionally EsxN-PPE68-EspA-TB8.4) immunoprotective Mtb antigens (rLmMtb9Ag) and evaluated them for safety, immunogenicity and efficacy as standalone vaccines in two mouse models and an outbred guinea pig model. In immunogenicity studies, rLmMtb5Ag administered subcutaneously induces significantly enhanced antigen-specific CD4+ and CD8+ T-cell responses in C57BL/6 and BALB/c mice, and rLmMtb9Ag induces antigen-specific CD4+ and CD8+ T-cell proliferation in guinea pigs. In efficacy studies, both rLmMtb5Ag and rLmMtb9Ag are safe and protect C57BL/6 and BALB/c mice and guinea pigs against aerosol challenge with highly virulent Mtb. Hence, multi-antigenic rLm vaccines hold promise as new vaccines against TB.

Atomic Structure of IglD Demonstrates Its Role as a Component of the Baseplate Complex of the Francisella Type VI Secretion System.

Francisella tularensis, a Tier 1 select agent of bioterrorism, contains a type VI secretion system (T6SS) encoded within the Francisella pathogenicity island (FPI), which is critical for its pathogenesis. Among the 18 proteins encoded by FPI is IglD, which is essential to Francisella's intracellular growth and virulence, but neither its location within T6SS nor its functional role has been established. Here, we present the cryoEM structure of IglD from Francisella novicida and show that the Francisella IglD forms a homotrimer that is structurally homologous to the T6SS baseplate protein TssK in Escherichia coli. Each IglD monomer consists of an N-terminal ß-sandwich domain, a 4-helix bundle domain, and a flexible C-terminal domain. While the overall folds of IglD and TssK are similar, the two structures differ in three aspects: the relative orientation between their ß-sandwich and the 4-helix bundle domains; two insertion loops present in TssK's ß-sandwich domain; and, consequently, a lack of subunit-subunit interaction between insertion loops in the IglD trimer. Phylogenetic analysis indicates that IglD is genetically remote from the TssK orthologs in other T6SSs. While the other components of the Francisella baseplate are unknown, we conducted pulldown assays showing IglJ interacts with IglD and IglH, pointing to a model wherein IglD, IglH, and IglJ form the baseplate of the Francisella T6SS. Alanine substitution mutagenesis further established that IglD's hydrophobic pocket in the N-terminal ß-sandwich domain interacts with two loops of IglJ, reminiscent of the TssK-TssG interaction. These results form a framework for understanding the hitherto unexplored Francisella T6SS baseplate. IMPORTANCE Francisella tularensis is a facultatively intracellular Gram-negative bacterium that causes the serious and potentially fatal zoonotic illness, tularemia. Because of its extraordinarily high infectivity and mortality to humans, especially when inhaled, F. tularensis is considered a potential bioterrorism agent and is classified as a Tier 1 select agent. The type VI secretion system (T6SS) encoded within the Francisella pathogenicity island (FPI) is critical to its pathogenesis, but its baseplate components are largely unknown. Here, we report the cryoEM structure of IglD from Francisella novicida and demonstrate its role as a component of the baseplate complex of the Francisella T6SS. We further show that IglD interacts with IglJ and IglH, and propose a model in which these proteins interact to form the Francisella T6SS baseplate. Elucidation of the structure and composition of the Francisella baseplate should facilitate the design of strategies to prevent and treat infections caused by F. tularensis.

Listeria-Vectored Multiantigenic Tuberculosis Vaccine Enhances Protective Immunity against Aerosol Challenge with Virulent Mycobacterium tuberculosis in BCG-Immunized C57BL/6 and BALB/c Mice.

Mycobacterium tuberculosis infects approximately one-third of the world's population, causing active tuberculosis (TB) in ~10 million people and death in ~1.5 million people annually. A potent vaccine is needed to boost the level of immunity conferred by the current Mycobacterium bovis BCG vaccine that provides moderate protection against childhood TB but variable protection against adult pulmonary TB. Previously, we developed a recombinant attenuated Listeria monocytogenes (rLm)-vectored M. tuberculosis vaccine expressing the M. tuberculosis 30-kDa major secretory protein (r30/Ag85B), recombinant attenuated L. monocytogenes ΔactA ΔinlB prfA*30 (rLm30), and showed that boosting BCG-primed mice and guinea pigs with rLm30 enhances immunoprotection against challenge with aerosolized M. tuberculosis Erdman strain. To broaden the antigen repertoire and robustness of rLm30, we constructed 16 recombinant attenuated L. monocytogenes vaccine candidates expressing 3, 4, or 5 among 15 selected M. tuberculosis antigens, verified their protein expression, genetic stability, and growth kinetics in macrophages, and evaluated them for capacity to boost protective efficacy in BCG-primed mice. We found that boosting BCG-primed C57BL/6 and BALB/c mice with recombinant attenuated L. monocytogenes multiantigenic M. tuberculosis vaccines, especially the rLm5Ag(30) vaccine expressing a fusion protein of 23.5/Mpt64, TB10.4/EsxH, ESAT6/EsxA, CFP10/EsxB, and r30, enhances BCG-induced protective immunity against M. tuberculosis aerosol challenge. In immunogenicity studies, rLm5Ag(30) strongly boosts M. tuberculosis antigen-specific CD4-positive (CD4+) and CD8+ T cell-mediated TH1-type immune responses in the spleens and lungs of BCG-primed C57BL/6 mice but does so only weakly in BCG-primed BALB/c mice. Hence, rLm5Ag(30) boosts BCG-primed immunoprotection against M. tuberculosis aerosol challenge in both C57BL/6 and BALB/c mice despite major differences in the magnitude of the vaccine-induced Th1 response in these mouse strains. Given the consistency with which recombinant attenuated L. monocytogenes vaccines expressing the 5 M. tuberculosis antigens in rLm5Ag(30) are able to boost the already high level of protection conferred by BCG alone in two rigorous mouse models of pulmonary TB and the broad CD4+ and CD8+ T cell immunity induced by rLm5Ag(30), this vaccine holds considerable promise as a new vaccine to combat the TB pandemic, especially for the majority of the world's population immunized with BCG in infancy. IMPORTANCE TB, one of the world's most important infectious diseases, afflicts approximately 10 million people and kills approximately 1.5 million people annually. The current vaccine, BCG, developed over a century ago, has been administered to about 5 billion people, mostly in infancy, but is only modestly protective. Hence, a vaccine is urgently needed to boost the level of protection afforded by BCG. Herein, we describe a safe potent live vaccine that utilizes as a vector an attenuated strain of Listeria monocytogenes, a bacterium that mimics the intracellular lifestyle of Mycobacterium tuberculosis, the causative agent of TB. The vaccine produces multiple immunologically protective proteins of M. tuberculosis. In two mouse models of pulmonary TB, the vaccine boosts the level of protection afforded by BCG. Thus, this vaccine holds considerable promise as a new vaccine to combat the TB pandemic, especially for the majority of the world's population immunized with BCG.

Replicating bacterium-vectored vaccine expressing SARS-CoV-2 Membrane and Nucleocapsid proteins protects against severe COVID-19-like disease in hamsters.

To generate an inexpensive readily manufactured COVID-19 vaccine, we employed the LVS ΔcapB vector platform, previously used to generate potent candidate vaccines against Select Agent diseases tularemia, anthrax, plague, and melioidosis. Vaccines expressing SARS-CoV-2 structural proteins are constructed using the LVS ΔcapB vector, a highly attenuated replicating intracellular bacterium, and evaluated for efficacy in golden Syrian hamsters, which develop severe COVID-19-like disease. Hamsters immunized intradermally or intranasally with a vaccine co-expressing the Membrane and Nucleocapsid proteins and challenged 5 weeks later with a high dose of SARS-CoV-2 are protected against severe weight loss and lung pathology and show reduced viral loads in the oropharynx and lungs. Protection correlates with anti-Nucleocapsid antibody. This potent vaccine should be safe; inexpensive; easily manufactured, stored, and distributed; and given the high homology between Membrane and Nucleocapsid proteins of SARS-CoV and SARS-CoV-2, potentially serve as a universal vaccine against the SARS subset of pandemic causing ß-coronaviruses.

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.

Replicating bacterium-vectored vaccine expressing SARS-CoV-2 Membrane and Nucleocapsid proteins protects against severe COVID-19 disease in hamsters.

An inexpensive readily manufactured COVID-19 vaccine that protects against severe disease is needed to combat the pandemic. We have employed the LVS Δ capB vector platform, previously used successfully to generate potent vaccines against the Select Agents of tularemia, anthrax, plague, and melioidosis, to generate a COVID-19 vaccine. The LVS Δ capB vector, a replicating intracellular bacterium, is a highly attenuated derivative of a tularemia vaccine (LVS) previously administered to millions of people. We generated vaccines expressing SARS-CoV-2 structural proteins and evaluated them for efficacy in the golden Syrian hamster, which develops severe COVID-19 disease. Hamsters immunized intradermally or intranasally with a vaccine co-expressing the Membrane (M) and Nucleocapsid (N) proteins, then challenged 5-weeks later with a high dose of SARS-CoV-2, were protected against severe weight loss and lung pathology and had reduced viral loads in the oropharynx and lungs. Protection by the vaccine, which induces murine N-specific interferon-gamma secreting T cells, was highly correlated with pre-challenge serum anti-N TH1-biased IgG. This potent vaccine against severe COVID-19 should be safe and easily manufactured, stored, and distributed, and given the high homology between MN proteins of SARS-CoV and SARS-CoV-2, has potential as a universal vaccine against the SARS subset of pandemic causing ß-coronaviruses.

Atomic Structure of the Francisella T6SS Central Spike Reveals a Unique α-Helical Lid and a Putative Cargo.

Francisella bacteria rely on a phylogenetically distinct type VI secretion system (T6SS) to escape host phagosomes and cause the fatal disease tularemia, but the structural and molecular mechanisms involved are unknown. Here we report the atomic structure of the Francisella T6SS central spike complex, obtained by cryo-electron microscopy. Our structural and functional studies demonstrate that, unlike the single-protein spike composition of other T6SS subtypes, Francisella T6SS's central spike is formed by two proteins, PdpA and VgrG, akin to T4-bacteriophage gp27 and gp5, respectively, and that PdpA has unique characteristics, including a putative cargo within its cavity and an N-terminal helical lid. Structure-guided mutagenesis demonstrates that the PdpA N-terminal lid and C-terminal spike are essential to Francisella T6SS function. PdpA is thus both an adaptor, connecting VgrG to the tube, and a likely carrier of secreted cargo. These findings are important to understanding Francisella pathogenicity and designing therapeutics to combat tularemia.

Artificial intelligence enabled parabolic response surface platform identifies ultra-rapid near-universal TB drug treatment regimens comprising approved drugs.

BACKGROUND: Shorter, more effective treatments for tuberculosis (TB) are urgently needed. While many TB drugs are available, identification of the best regimens is challenging because of the large number of possible drug-dose combinations. We have found consistently that responses of cells or whole animals to drug-dose stimulations fit a parabolic response surface (PRS), allowing us to identify and optimize the best drug combinations by testing only a small fraction of the total search space. Previously, we used PRS methodology to identify three regimens (PRS Regimens I-III) that in murine models are much more effective than the standard regimen used to treat TB. However, PRS Regimens I and II are unsuitable for treating drug-resistant TB and PRS Regimen III includes an experimental drug. Here, we use PRS methodology to identify from an expanded pool of drugs new highly effective near-universal drug regimens comprising only approved drugs. METHODS AND FINDINGS: We evaluated combinations of 15 different drugs in a human macrophage TB model and identified the most promising 4-drug combinations. We then tested 14 of these combinations in Mycobacterium tuberculosis-infected BALB/c mice and chose for PRS dose optimization and further study the two most potent regimens, designated PRS Regimens IV and V, consisting of clofazimine (CFZ), bedaquiline (BDQ), pyrazinamide (PZA), and either amoxicillin/clavulanate (AC) or delamanid (DLM), respectively. We then evaluated the efficacy in mice of optimized PRS Regimens IV and V, as well as a 3-drug regimen, PRS Regimen VI (CFZ, BDQ, and PZA), and compared their efficacy to PRS Regimen III (CFZ, BDQ, PZA, and SQ109), previously shown to reduce the time to achieve relapse-free cure in mice by 80% compared with the Standard Regimen (isoniazid, rifampicin, PZA, and ethambutol). Efficacy measurements included early bactericidal activity, time to lung sterilization, and time to relapse-free cure. PRS Regimens III-VI all rapidly sterilized the lungs and achieved relapse-free cure in 3 weeks (PRS Regimens III, V, and VI) or 5 weeks (PRS Regimen IV). In contrast, mice treated with the Standard Regimen still had high numbers of bacteria in their lungs after 6-weeks treatment and none achieved relapse-free cure in this time-period. CONCLUSIONS: We have identified three new regimens that rapidly sterilize the lungs of mice and dramatically shorten the time required to achieve relapse-free cure. All mouse drug doses in these regimens extrapolate to doses that are readily achievable in humans. Because PRS Regimens IV and V contain only one first line drug (PZA) and exclude fluoroquinolones and aminoglycosides, they should be effective against most TB cases that are multidrug resistant (MDR-TB) and many that are extensively drug-resistant (XDR-TB). Hence, these regimens have potential to shorten dramatically the time required for treatment of both drug-sensitive and drug-resistant TB. If clinical trials confirm that these regimens dramatically shorten the time required to achieve relapse-free cure in humans, then this radically shortened treatment has the potential to improve treatment compliance, decrease the emergence of drug resistance, and decrease the healthcare burden of treating both drug-sensitive and drug-resistant TB.

Nanomachines and Other Caps on Mesoporous Silica Nanoparticles for Drug Delivery.

Mesoporous silica nanoparticles (MSNs) are delivery vehicles that can carry cargo molecules and release them on command. The particles used in the applications reported in this Account are around 100 nm in diameter (about the size of a virus) and contain 2.5 nm tubular pores with a total volume of about 1 cm3/g. For the biomedical applications discussed here, the cargo is trapped in the pores until the particles are stimulated to release it. The challenges are to get the particles to the site of a disease and then to deliver the cargo on command. We describe methods to do both, and we illustrate the applicability of the particles to cure cancer and intracellular infectious disease. Our first steps were to design multifunctional nanoparticles with properties that allow them to carry and deliver hydrophobic drugs. Many important pharmaceuticals are hydrophobic and cannot reach the diseased sites by themselves. We describe how we modified MSNs to make them dispersible, imagable, and targetable and discuss in vitro studies. We then present examples of surface modifications that allow them to deliver large molecules such as siRNA. In vivo studies of siRNA delivery to treat triple-negative breast and ovarian cancers are presented. The next steps are to attach nanomachines and other types of caps that trap drug molecules but release them when stimulated. We describe nanomachines that respond autonomously (without human intervention) to stimuli specific to disease sites. A versatile type of machine is a nanovalve that is closed at neutral (blood) pH but opens upon acidification that occurs in endolysosomes of cancer cells. Another type of machine, a snap-top cap, is stimulated by reducing agents such as glutathione in the cytosol of cells. Both of these platforms were studied in vitro to deliver antibiotics to infected macrophages and in vivo to cure and kill the intracellular bacteria M. tuberculosis and F. tularensis. The latter is a tier 1 select agent of bioterrorism. Finally, we describe nanomachines for drug delivery that are controlled by externally administered light and magnetic fields. A futuristic dream for nanotherapy is the ability to control a nano-object everywhere in the body. Magnetic fields penetrate completely and have spatial selectivity governed by the size of the field-producing coil. We describe how to control nanovalves with alternating magnetic fields (AMFs) and superparamagnetic cores inside the MSNs. The AMF heats the cores, and temperature-sensitive caps release the cargo. In vitro studies demonstrate dose control of the therapeutic to cause apoptosis without overheating the cells. Nanocarriers have great promise for therapeutic applications, and MSNs that can carry drugs to the site of a disease to produce a high local concentration without premature release and off-target damage may have the capability of realizing this goal.

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.

Ultra-rapid near universal TB drug regimen identified via parabolic response surface platform cures mice of both conventional and high susceptibility.

As current treatment of tuberculosis is burdensomely long, provoking non-adherence and drug resistance, effective short-course treatments are needed. Using the output-driven parabolic response surface (PRS) platform, we have identified drug regimens that treat tuberculosis more rapidly in mice than the current Standard Regimen used in humans. We show that PRS Regimen III, comprising clofazimine, SQ109, bedaquiline and pyrazinamide, rapidly sterilizes the lung both in conventionally studied BALB/c mice and in C3HeB/FeJ mice, highly susceptible mice that develop massive necrotic granulomatous lung lesions akin to those in humans, achieving relapse-free cure in only 4 weeks (p<0.0001 versus Standard Regimen). In contrast, the Standard Regimen required 16 weeks to attain lung culture negative status and 20 weeks to achieve relapse-free cure. Thus, PRS Regimen III dramatically cuts by ~80% the time to relapse-free cure in mouse tuberculosis models. PRS Regimen III, with three nonstandard drugs, can potentially treat both drug-sensitive and most drug-resistant tuberculosis.

Facile Strategy Enabling Both High Loading and High Release Amounts of the Water-Insoluble Drug Clofazimine Using Mesoporous Silica Nanoparticles.

The use of nanocarriers to deliver poorly soluble drugs to the sites of diseases is an attractive and general method, and mesoporous silica nanoparticles (MSNs) are increasingly being used as carriers. However, both loading a large amount of drugs into the pores and still being able to release the drug is a challenge. In this paper, we demonstrate a general strategy based on a companion molecule that chaperones the drug into the pores and also aids it in escaping. A common related strategy is to use a miscible co-solvent dimethyl sulfoxide (DMSO), but although loading may be efficient in DMSO, this co-solvent frequently diffuses into an aqueous environment, leaving the drug behind. We demonstrate the method by using acetophenone (AP), an FDA-approved food additive as the chaperone for clofazimine (CFZ), a water-insoluble antibiotic used to treat leprosy and multidrug-resistant tuberculosis. AP enables a high amount of CFZ cargo into the MSNs and also carries CFZ cargo out from the MSNs effectively when they are in an aqueous biorelevant environment. The amount of loading and the CFZ release efficiency from MSNs were optimized; 4.5 times more CFZ was loaded in MSNs with AP than that with DMSO and 2300 times more CFZ was released than that without the assistance of the AP. In vitro treatment of macrophages infected by Mycobacterium tuberculosis with the optimized CFZ-loaded MSNs killed the bacteria in the cells in a dose-dependent manner. These studies demonstrate a highly efficient method for loading nanoparticles with water-insoluble drug molecules and the efficacy of the nanoparticles in delivering drugs into eukaryotic cells in aqueous media.

Live Attenuated Tularemia Vaccines for Protection Against Respiratory Challenge With Virulent F. tularensis subsp. tularensis.

Francisella tularensis is the causative agent of tularemia and a Tier I bioterrorism agent. In the 1900s, several vaccines were developed against tularemia including the killed "Foshay" vaccine, subunit vaccines comprising F. tularensis protein(s) or lipoproteins(s) in an adjuvant formulation, and the F. tularensis Live Vaccine Strain (LVS); none were licensed in the U.S.A. or European Union. The LVS vaccine retains toxicity in humans and animals-especially mice-but has demonstrated efficacy in humans, and thus serves as the current gold standard for vaccine efficacy studies. The U.S.A. 2001 anthrax bioterrorism attack spawned renewed interest in vaccines against potential biowarfare agents including F. tularensis. Since live attenuated-but not killed or subunit-vaccines have shown promising efficacy and since vaccine efficacy against respiratory challenge with less virulent subspecies holarctica or F. novicida, or against non-respiratory challenge with virulent subsp. tularensis (Type A) does not reliably predict vaccine efficacy against respiratory challenge with virulent subsp. tularensis, the route of transmission and species of greatest concern in a bioterrorist attack, in this review, we focus on live attenuated tularemia vaccine candidates tested against respiratory challenge with virulent Type A strains, including homologous vaccines derived from mutants of subsp. holarctica, F. novicida, and subsp. tularensis, and heterologous vaccines developed using viral or bacterial vectors to express F. tularensis immunoprotective antigens. We compare the virulence and efficacy of these vaccine candidates with that of LVS and discuss factors that can significantly impact the development and evaluation of live attenuated tularemia vaccines. Several vaccines meet what we would consider the minimum criteria for vaccines to go forward into clinical development-safety greater than LVS and efficacy at least as great as LVS, and of these, several meet the higher standard of having efficacy ≥LVS in the demanding mouse model of tularemia. These latter include LVS with deletions in purMCD, sodBFt , capB or wzy; LVS ΔcapB that also overexpresses Type VI Secretion System (T6SS) proteins; FSC200 with a deletion in clpB; the single deletional purMCD mutant of F. tularensis SCHU S4, and a heterologous prime-boost vaccine comprising LVS ΔcapB and Listeria monocytogenes expressing T6SS proteins.

Single vector platform vaccine protects against lethal respiratory challenge with Tier 1 select agents of anthrax, plague, and tularemia.

Bacillus anthracis, Yersinia pestis, and Francisella tularensis are the causative agents of Tier 1 Select Agents anthrax, plague, and tularemia, respectively. Currently, there are no licensed vaccines against plague and tularemia and the licensed anthrax vaccine is suboptimal. Here we report F. tularensis LVS ΔcapB (Live Vaccine Strain with a deletion in capB)- and attenuated multi-deletional Listeria monocytogenes (Lm)-vectored vaccines against all three aforementioned pathogens. We show that LVS ΔcapB- and Lm-vectored vaccines express recombinant B. anthracis, Y. pestis, and F. tularensis immunoprotective antigens in broth and in macrophage-like cells and are non-toxic in mice. Homologous priming-boosting with the LVS ΔcapB-vectored vaccines induces potent antigen-specific humoral and T-cell-mediated immune responses and potent protective immunity against lethal respiratory challenge with all three pathogens. Protection against anthrax was far superior to that obtained with the licensed AVA vaccine and protection against tularemia was comparable to or greater than that obtained with the toxic and unlicensed LVS vaccine. Heterologous priming-boosting with LVS ΔcapB- and Lm-vectored B. anthracis and Y. pestis vaccines also induced potent protective immunity against lethal respiratory challenge with B. anthracis and Y. pestis. The single vaccine platform, especially the LVS ΔcapB-vectored vaccine platform, can be extended readily to other pathogens.

The Francisella Type VI Secretion System.

Francisella tularensisis subsp. tularensis is an intracellular bacterial pathogen and the causative agent of the life-threatening zoonotic disease tularemia. The Francisella Pathogenicity Island encodes a large secretion apparatus, known as a Type VI Secretion System (T6SS), which is essential for Francisella to escape from its phagosome and multiply within host macrophages and to cause disease in animals. The T6SS, found in one-quarter of Gram-negative bacteria including many highly pathogenic ones, is a recently discovered secretion system that is not yet fully understood. Nevertheless, there have been remarkable advances in our understanding of the structure, composition, and function of T6SSs of several bacteria in the past few years. The system operates like an inside-out headless contractile phage that is anchored to the bacterial membrane via a baseplate and membrane complex. The system injects effector molecules across the inner and outer bacterial membrane and into host prokaryotic or eukaryotic targets to kill, intoxicate, or in the case of Francisella, hijack the target cell. Recent advances include an atomic model of the contractile sheath, insights into the mechanics of sheath contraction, the composition of the baseplate and membrane complex, the process of assembly of the apparatus, and identification of numerous effector molecules and activities. While Francisella T6SS appears to be an outlier among T6SSs, with limited or no sequence homology with other systems, its structure and organization are strikingly similar to other systems. Nevertheless, we have only scratched the surface in uncovering the mysteries of the Francisella T6SS, and there are numerous questions that remain to be answered.