Evaluation of repRNA vaccine for induction and in utero transfer of maternal antibodies in a pregnant rabbit model.

Mother-to-child transmission is a major route for infections in newborns. Vaccination in mothers to leverage the maternal immune system is a promising approach to vertically transfer protective immunity. During infectious disease outbreaks, such as the 2016 Zika virus (ZIKV) outbreak, rapid availability of vaccines can prove critical in reducing widespread disease burden. The recent successes of mRNA vaccines support their evaluation in pregnant animal models to justify their use in neonatal settings. Here we evaluated immunogenicity of self-amplifying replicon (repRNA) vaccines, delivered with our clinical-stage LION nanoparticle formulation, in pregnant rabbits using ZIKV and HIV-1 as model disease targets. We showed that LION/repRNA vaccines induced robust antigen-specific antibody responses in adult pregnant rabbits that passively transferred to newborn kits in utero. Using a matrixed study design, we further elucidate the effect of vaccination in kits on the presence of pre-existing maternal antibodies. Our findings showed that timing of maternal vaccination is critical in maximizing in utero antibody transfer, and subsequent vaccination in newborns maintained elevated antibody levels compared with no vaccination. Overall, our results support further development of the LION/repRNA vaccine platform for maternal and neonatal settings.

A localizing nanocarrier formulation enables multi-target immune responses to multivalent replicating RNA with limited systemic inflammation.

RNA vaccines possess significant clinical promise in counteracting human diseases caused by infectious or cancerous threats. Self-amplifying replicon RNA (repRNA) has been thought to offer the potential for enhanced potency and dose sparing. However, repRNA is a potent trigger of innate immune responses in vivo, which can cause reduced transgene expression and dose-limiting reactogenicity, as highlighted by recent clinical trials. Here, we report that multivalent repRNA vaccination, necessitating higher doses of total RNA, could be safely achieved in mice by delivering multiple repRNAs with a localizing cationic nanocarrier formulation (LION). Intramuscular delivery of multivalent repRNA by LION resulted in localized biodistribution accompanied by significantly upregulated local innate immune responses and the induction of antigen-specific adaptive immune responses in the absence of systemic inflammatory responses. In contrast, repRNA delivered by lipid nanoparticles (LNPs) showed generalized biodistribution, a systemic inflammatory state, an increased body weight loss, and failed to induce neutralizing antibody responses in a multivalent composition. These findings suggest that in vivo delivery of repRNA by LION is a platform technology for safe and effective multivalent vaccination through mechanisms distinct from LNP-formulated repRNA vaccines.

A Structure-Function-Inhibition Analysis of the Pseudomonas aeruginosa Type III Secretion Needle Protein PscF.

The Pseudomonas aeruginosa type III secretion system (T3SS) needle comprised of multiple PscF subunits is essential for the translocation of effector toxins into human cells, facilitating the establishment and dissemination of infection. Mutations in the pscF gene provide resistance to the phenoxyacetamide (PhA) series of T3SS inhibitory chemical probes. To better understand PscF functions and interactions with PhA, alleles of pscF with 71 single mutations altering 49 of the 85 residues of the encoded protein were evaluated for their effects on T3SS phenotypes. Of these, 37% eliminated and 63% maintained secretion, with representatives of both evenly distributed across the entire protein. Mutations in 14 codons conferred a degree of PhA resistance without eliminating secretion, and all but one were in the alpha-helical C-terminal 25% of PscF. PhA-resistant mutants exhibited no cross-resistance to two T3SS inhibitors with different chemical scaffolds. Two mutations caused constitutive T3SS secretion. The pscF allele at its native locus, whether wild type (WT), constitutive, or PhA resistant, was dominant over other pscF alleles expressed from nonnative loci and promoters, but mixed phenotypes were observed in chromosomal ΔpscF strains with both WT and mutant alleles at nonnative loci. Some PhA-resistant mutants exhibited reduced translocation efficiency that was improved in a PhA dose-dependent manner, suggesting that PhA can bind to those resistant needles. In summary, these results are consistent with a direct interaction between PhA inhibitors and the T3SS needle, suggest a mechanism of blocking conformational changes, and demonstrate that PscF affects T3SS regulation, as well as carrying out secretion and translocation.IMPORTANCEP. aeruginosa effector toxin translocation into host innate immune cells is critical for the establishment and dissemination of P. aeruginosa infections. The medical need for new anti-P. aeruginosa agents is evident by the fact that P. aeruginosa ventilator-associated pneumonia is associated with a high mortality rate (40 to 69%) and recurs in >30% of patients, even with standard-of-care antibiotic therapy. The results described here confirm roles for the PscF needle in T3SS secretion and translocation and suggest that it affects regulation, possibly by interaction with T3SS regulatory proteins. The results also support a model of direct interaction of the needle with PhA and suggest that, with further development, members of the PhA series may prove useful as drugs for P. aeruginosa infection.

Novel MenA Inhibitors Are Bactericidal against Mycobacterium tuberculosis and Synergize with Electron Transport Chain Inhibitors.

Mycobacterium tuberculosis is the leading cause of morbidity and death resulting from infectious disease worldwide. The incredible disease burden, combined with the long course of drug treatment and an increasing incidence of antimicrobial resistance among M. tuberculosis isolates, necessitates novel drugs and drug targets for treatment of this deadly pathogen. Recent work has produced several promising clinical candidates targeting components of the electron transport chain (ETC) of M. tuberculosis, highlighting this pathway's potential as a drug target. Menaquinone is an essential component of the M. tuberculosis ETC, as it functions to shuttle electrons through the ETC to produce the electrochemical gradient required for ATP production for the cell. We show that inhibitors of MenA, a component of the menaquinone biosynthetic pathway, are highly active against M. tuberculosis MenA inhibitors are bactericidal against M. tuberculosis under both replicating and nonreplicating conditions, with 10-fold higher bactericidal activity against nutrient-starved bacteria than against replicating cultures. MenA inhibitors have enhanced activity in combination with bedaquiline, clofazimine, and inhibitors of QcrB, a component of the cytochrome bc1 oxidase. Together, these data support MenA as a viable target for drug treatment against M. tuberculosis MenA inhibitors not only kill M. tuberculosis in a variety of physiological states but also show enhanced activity in combination with ETC inhibitors in various stages of clinical trial testing.

Combinations of Respiratory Chain Inhibitors Have Enhanced Bactericidal Activity against Mycobacterium tuberculosis.

As an obligate aerobe, Mycobacterium tuberculosis uses its electron transport chain (ETC) to produce energy via oxidative phosphorylation. This pathway has recently garnered a lot of attention and is a target for several new antimycobacterials. We tested the respiratory adaptation of M. tuberculosis to phenoxyalkylbenzimidazoles (PABs), compounds proposed to target QcrB, a component of the cytochrome bc1 complex. We show that M. tuberculosis is able to reroute its ETC to provide temporary resistance to PABs. However, combination treatment of PAB with agents targeting other components of the electron transport chain overcomes this respiratory flexibility. PAB in combination with clofazimine resulted in synergistic killing of M. tuberculosis under both replicating and nonreplicating conditions. PABs in combination with bedaquiline demonstrated antagonism at early time points, particularly under nonreplicating conditions. However, this antagonistic effect disappeared within 3 weeks, when PAB-BDQ combinations became highly bactericidal; in some cases, they were better than either drug alone. This study highlights the potential for combination treatment targeting the ETC and supports the development of PABs as part of a novel drug regimen against M. tuberculosis.

Impact of Type III Secretion Effectors and of Phenoxyacetamide Inhibitors of Type III Secretion on Abscess Formation in a Mouse Model of Pseudomonas aeruginosa Infection.

Pseudomonas aeruginosa is a leading cause of intra-abdominal infections, wound infections, and community-acquired folliculitis, each of which may involve macro- or microabscess formation. The rising incidence of multidrug resistance among P. aeruginosa isolates has increased both the economic burden and the morbidity and mortality associated with P. aeruginosa disease and necessitates a search for novel therapeutics. Previous work from our group detailed novel phenoxyacetamide inhibitors that block type III secretion and injection into host cells in vitro In this study, we used a mouse model of P. aeruginosa abscess formation to test the in vivo efficacy of these compounds against the P. aeruginosa type III secretion system (T3SS). Bacteria used the T3SS to intoxicate infiltrating neutrophils to establish abscesses. Despite this antagonism, sufficient numbers of functioning neutrophils remained for proper containment of the abscesses, as neutrophil depletion resulted in an increased abscess size, the formation of dermonecrotic lesions on the skin, and the dissemination of P. aeruginosa to internal organs. Consistent with the specificity of the T3SS-neutrophil interaction, P. aeruginosa bacteria lacking a functional T3SS were fully capable of causing abscesses in a neutropenic host. Phenoxyacetamide inhibitors attenuated abscess formation and aided in the immune clearance of the bacteria. Finally, a P. aeruginosa strain resistant to the phenoxyacetamide compound was fully capable of causing abscess formation even in the presence of the T3SS inhibitors. Together, our results further define the role of type III secretion in murine abscess formation and demonstrate the in vivo efficacy of phenoxyacetamide inhibitors in P. aeruginosa infection.

Pseudomonas aeruginosa: breaking down barriers.

Many bacterial pathogens have evolved ingenious ways to escape from the lung during pneumonia to cause bacteremia. Unfortunately, the clinical consequences of this spread to the bloodstream are frequently dire. It is therefore important to understand the molecular mechanisms used by pathogens to breach the lung barrier. We have recently shown that Pseudomonas aeruginosa, one of the leading causes of hospital-acquired pneumonia, utilizes the type III secretion system effector ExoS to intoxicate pulmonary epithelial cells. Injection of these cells leads to localized disruption of the pulmonary-vascular barrier and dissemination of P. aeruginosa to the bloodstream. We put these data in the context of previous studies to provide a holistic model of P. aeruginosa dissemination from the lung. Finally, we compare P. aeruginosa dissemination to that of other bacteria to highlight the complexity of bacterial pneumonia. Although respiratory pathogens use distinct and intricate strategies to escape from the lungs, a thorough understanding of these processes can lay the foundation for new therapeutic approaches for bacterial pneumonia.

Targeting Staphylococcus aureus α-toxin as a novel approach to reduce severity of recurrent skin and soft-tissue infections.

Staphyococcus aureus frequently causes recurrent skin and soft-tissue infection (SSTI). In the pediatric population, elevated serum antibody targeting S. aureus α-toxin is correlated with a reduced incidence of recurrent SSTI. Using a novel model of recurrent SSTI, we demonstrated that expression of α-toxin during primary infection increases the severity of recurrent disease. Antagonism of α-toxin by either a dominant-negative toxin mutant or a small molecule inhibitor of the toxin receptor ADAM10 during primary infection reduces reinfection abscess severity. Early neutralization of α-toxin activity during S. aureus SSTI therefore offers a new therapeutic strategy to mitigate primary and recurrent disease.

The psmα locus regulates production of Staphylococcus aureus alpha-toxin during infection.

Staphylococcus aureus is a leading cause of human bacterial infection, causing a wide spectrum of disease ranging from skin and soft tissue infections to life-threatening pneumonia and sepsis. S. aureus toxins play an essential role in disease pathogenesis, contributing to both immunomodulation and host tissue injury. Prominent among these toxins are the membrane-active pore-forming cytolysin alpha-toxin (Hla) and the amphipathic α-helical phenol-soluble modulin (PSM) peptides. As deletion of either the hla or psm locus leads to a phenotypically similar virulence defect in skin and soft tissue infection, we sought to determine the relative contribution of each locus to disease pathogenesis. Here we show that production of Hla can be modulated by PSM expression. An S. aureus mutant lacking PSM expression exhibits a transcriptional delay in hla mRNA production and therefore fails to secrete normal levels of Hla at early phases of growth. This leads to attenuation of virulence in vitro and in murine skin and lung models of infection, correlating with reduced recovery of Hla from host tissues. Production of Hla and restoration of staphylococcal virulence can be achieved in the psm mutant by plasmid-driven overexpression of hla. Our study suggests the coordinated action of Hla and PSMs in host tissue during early pathogenesis, confirming a major role for Hla in epithelial injury during S. aureus infection. These findings highlight the possibility that therapeutics targeting PSM production may simultaneously prevent Hla-mediated tissue injury.

Protective interplay: Mycobacterium tuberculosis diminishes SARS-CoV-2 severity through innate immune priming.

At the beginning of the COVID-19 pandemic those with underlying chronic lung conditions, including tuberculosis (TB), were hypothesized to be at higher risk of severe COVID-19 disease. However, there is inconclusive clinical and preclinical data to confirm the specific risk SARS-CoV-2 poses for the millions of individuals infected with Mycobacterium tuberculosis (M.tb). We and others have found that compared to singly infected mice, mice co-infected with M.tb and SARS-CoV-2 leads to reduced SARS-CoV-2 severity compared to mice infected with SARS-CoV-2 alone. Consequently, there is a large interest in identifying the molecular mechanisms responsible for the reduced SARS-CoV-2 infection severity observed in M.tb and SARS-CoV-2 co-infection. To address this, we conducted a comprehensive characterization of a co-infection model and performed mechanistic in vitro modeling to dynamically assess how the innate immune response induced by M.tb restricts viral replication. Our study has successfully identified several cytokines that induce the upregulation of anti-viral genes in lung epithelial cells, thereby providing protection prior to challenge with SARS-CoV-2. In conclusion, our study offers a comprehensive understanding of the key pathways induced by an existing bacterial infection that effectively restricts SARS-CoV-2 activity and identifies candidate therapeutic targets for SARS-CoV-2 infection.

Preparation Strategies of the Anti-Mycobacterial Drug Bedaquiline for Intrapulmonary Routes of Administration.

Mycobacterium tuberculosis (M.tb) has infected one-quarter of the world's population and led to the deaths of 1.6 million individuals in 2021 according to estimates from the World Health Organization. The rise in prevalence of multidrug-resistant and extensively drug-resistant M.tb strains coupled with insufficient therapies to treat such strains has motivated the development of more effective treatments and/or delivery modalities. Bedaquiline, a diarylquinoline antimycobacterial agent, effectively targets mycobacterial ATP synthase but may lead to systemic complications upon oral delivery. Targeted delivery of bedaquiline to the lungs represents an alternative strategy to harness the sterilizing benefits of the drug against M.tb while mitigating off-target side effects. Two pulmonary delivery modalities were developed herein, including dry powder inhalation and liquid instillation. Despite bedaquiline's poor water solubility, spray drying was performed in predominantly aqueous conditions (≥80%) to avoid a closed-loop, inert system. Aerosols of spray-dried bedaquiline with L-leucine excipient outperformed spray-dried bedaquiline alone, demonstrating superior fine particle fraction metrics (~89% of the emitted dose below <5 µm), suitable for inhalation therapies. Furthermore, the use of a 2-hydroxypropyl-ß-cyclodextrin excipient allowed a molecular dispersion of bedaquiline in an aqueous solution for liquid instillation. Both delivery modalities were successfully administered to Hartley guinea pigs for pharmacokinetic analysis and were well-tolerated by the animals. Intrapulmonary liquid delivery of bedaquiline led to adequate serum absorption and appropriate peak serum concentrations of the drug. The liquid formulation was superior in systemic uptake compared to the powder formulation. The predominant route via which M.tb bacilli enter the body is aerosol droplets that are deposited onto airway surfaces. For this reason, we believe that further studies should focus on inhalation or intrapulmonary therapies that target the site of entry and primary site of infection for M.tb.

COVID-19 booster vaccination during pregnancy enhances maternal binding and neutralizing antibody responses and transplacental antibody transfer to the newborn.

The immune response to COVID-19 booster vaccinations during pregnancy for mothers and their newborns and the functional response of vaccine-induced antibodies against Omicron variants are not well characterized. We conducted a prospective, multicenter cohort study of participants vaccinated during pregnancy with primary or booster mRNA COVID-19 vaccines from July 2021 to January 2022 at 9 academic sites. We determined SARS-CoV-2 binding and live virus and pseudovirus neutralizing antibody (nAb) titers pre- and post-vaccination, and at delivery for both maternal and infant participants. Immune responses to ancestral and Omicron BA.1 SARS-CoV-2 strains were compared between primary and booster vaccine recipients in maternal sera at delivery and in cord blood, after adjusting for days since last vaccination. A total of 240 participants received either Pfizer or Moderna mRNA vaccine during pregnancy (primary 2-dose series: 167; booster dose: 73). Booster vaccination resulted in significantly higher binding and nAb titers, including to the Omicron BA.1 variant, in maternal serum at delivery and in cord blood compared to a primary 2-dose series (range 0.44-0.88 log10 higher, p < 0.0001 for all comparisons). Live virus nAb to Omicron BA.1 were present at delivery in 9 % (GMT ID50 12.7) of Pfizer and 22 % (GMT ID50 14.7) of Moderna primary series recipients, and in 73 % (GMT ID50 60.2) of mRNA boosted participants (p < 0.0001), although titers were significantly lower than to the D614G strain. Transplacental antibody transfer was efficient for all regimens with median transfer ratio range: 1.55-1.77 for IgG, 1.00-1.78 for live virus nAb and 1.79-2.36 for pseudovirus nAb. COVID-19 mRNA vaccination during pregnancy elicited robust immune responses in mothers and efficient transplacental antibody transfer to the newborn. A booster dose during pregnancy significantly increased maternal and cord blood binding and neutralizing antibody levels, including against Omicron BA.1. Findings support the use of a booster dose of COVID-19 vaccine during pregnancy.

Characterizing in vivo loss of virulence of an HN878 Mycobacterium tuberculosis isolate from a genetic duplication event.

The increase of global cases of drug resistant (DR) Mycobacterium tuberculosis (M.tb) is a serious problem for the tuberculosis research community and the goals to END TB by 2030. Due to the need for advancing and screening next generation therapeutics and vaccines, we aimed to design preclinical DR models of Beijing lineage M.tb HN878 strain in different mouse backgrounds. We found escalating sensitivities of morbidity due to low dose aerosol challenge (50-100 bacilli) in CB6F1, C57BL/6 and SWR mice, respectively. We also observed that pulmonary bacterial burden at morbidity endpoints correlated inversely with survival over time between mouse strains. Interestingly, with in vitro passaging and in the process of selecting individual DR mutant colonies, we observed a significant decrease in in vivo HN878 strain virulence, which correlated with the acquisition of a large genetic duplication. We confirmed that low passage infection stocks with no or low prevalence of the duplication, including stocks directly acquired from the BEI resources biorepository, retained virulence, measured by morbidity over time. These data help confirm previous reports and emphasize the importance of monitoring virulence and stock fidelity.

YopR impacts type III needle polymerization in Yersinia species.

A hallmark of Yersinia type III machines is the presence of needles extending from the bacterial surface. Needles perform two functions, serving as the conduits for the transport of effectors into immune cells but also acting as a sensor. The polymerized needle protein, YscF, is thought to perceive threshold levels of environmental calcium ions to trigger secretion. yopR (yscH) is a gene downstream of yscEFG, encoding the chaperones and principal building blocks of the needle. Here we investigated the contribution of YopR towards type III secretion and pathogenesis. Yersinia pestis KIM D27 mutants lacking yopR were defective for virulence in a mouse model of septicemic plague. yopR variants of Yersinia enterocolitica W22703 displayed a reduced ability to inject effectors into macrophages and required lower calcium concentrations to activate type III secretion than wild-type yersiniae. Furthermore, yopR mutants failed to assemble YscF into needle complexes and instead secreted YscF into the medium. These results imply that YopR may be involved in controlling the secretion of YscF, thereby impacting the assembly of type III machines. An alternative possibility, which YopR participates directly in the polymerization of YscF, seems less likely as YopR is not associated with purified needles.

Subunit vaccine protects against a clinical isolate of Mycobacterium avium in wild type and immunocompromised mouse models.

The nontuberculous mycobacteria (NTM) Mycobacterium avium is a clinically significant pathogen that can cause a wide range of maladies, including tuberculosis-like pulmonary disease. An immunocompromised host status, either genetically or acutely acquired, presents a large risk for progressive NTM infections. Due to this quietly emerging health threat, we evaluated the ability of a recombinant fusion protein ID91 combined with GLA-SE [glucopyranosyl lipid adjuvant, a toll like receptor 4 agonist formulated in an oil-in-water stable nano-emulsion] to confer protection in both C57BL/6 (wild type) and Beige (immunocompromised) mouse models. We optimized an aerosol challenge model using a clinical NTM isolate: M. avium 2-151 smt, observed bacterial growth kinetics, colony morphology, drug sensitivity and histopathology, characterized the influx of pulmonary immune cells, and confirmed the immunogenicity of ID91 in both mouse models. To determine prophylactic vaccine efficacy against this M. avium isolate, mice were immunized with either ID91 + GLA-SE or bacillus Calmette-Guérin (BCG). Immunocompromised Beige mice displayed a delayed influx of innate and adaptive immune cells resulting in a sustained and increased bacterial burden in the lungs and spleen compared to C57BL/6 mice. Importantly, both ID91 + GLA-SE and BCG vaccines significantly reduced pulmonary bacterial burden in both mouse strains. This work is a proof-of-concept study of subunit vaccine-induced protection against NTM.

Qualification of ELISA and neutralization methodologies to measure SARS-CoV-2 humoral immunity using human clinical samples.

In response to the SARS-CoV-2 pandemic many vaccines have been developed and evaluated in human clinical trials. The humoral immune response magnitude, composition and efficacy of neutralizing SARS-CoV-2 are essential endpoints for these trials. Robust assays that are reproducibly precise, linear, and specific for SARS-CoV-2 antigens would be beneficial for the vaccine pipeline. In this work we describe the methodologies and clinical qualification of three SARS-CoV-2 endpoint assays. We developed and qualified Endpoint titer ELISAs for total IgG, IgG1, IgG3, IgG4, IgM and IgA to evaluate the magnitude of specific responses to the trimeric spike (S) antigen and total IgG specific to the spike receptor binding domain (RBD) of SARS-CoV-2. We also qualified a pseudovirus neutralization assay which evaluates functional antibody titers capable of inhibiting the entry and replication of a lentivirus containing the Spike antigen of SARS-CoV-2. To complete the suite of assays we qualified a plaque reduction neutralization test (PRNT) methodology using the 2019-nCoV/USA-WA1/2020 isolate of SARS-CoV-2 to assess neutralizing titers of antibodies in plasma from normal healthy donors and convalescent COVID-19 individuals.

Qualification of ELISA and neutralization methodologies to measure SARS-CoV-2 humoral immunity using human clinical samples.

In response to the SARS-CoV-2 pandemic many vaccines have been developed and evaluated in human clinical trials. The humoral immune response magnitude, composition and efficacy of neutralizing SARS-CoV-2 are essential endpoints for these trials. Robust assays that are reproducibly precise, linear, and specific for SARS-CoV-2 antigens would be beneficial for the vaccine pipeline. In this work we describe the methodologies and clinical qualification of three SARS-CoV-2 endpoint assays. We developed and qualified Endpoint titer ELISAs for total IgG, IgG1, IgG3, IgG4, IgM and IgA to evaluate the magnitude of specific responses to the trimeric spike (S) antigen and total IgG specific to the spike receptor binding domain (RBD) of SARS-CoV-2. We also qualified a pseudovirus neutralization assay which evaluates functional antibody titers capable of inhibiting the entry and replication of a lentivirus containing the Spike antigen of SARS-CoV-2. To complete the suite of assays we qualified a plaque reduction neutralization test (PRNT) methodology using the 2019-nCoV/USA-WA1/2020 isolate of SARS-CoV-2 to assess neutralizing titers of antibodies in plasma from normal healthy donors and convalescent COVID-19 individuals.

Impassable YscP substrates and their impact on the Yersinia enterocolitica type III secretion pathway.

Yersinia type III machines secrete protein substrates across the bacterial envelope and, following assembly of their secretion needles, transport effector Yops into host cells. According to their destination during type III secretion, early, middle, and late secretion substrates can be distinguished; however, the signals and mechanisms whereby these proteins are recognized and transported by the secretion machine are not understood. Here, we examine several hybrids between secretion substrates and the impassable reporter protein glutathione S-transferase (GST). YscP-GST and YopR-GST blocked type III secretion; however, YscF-, YopD-, YopN-, and LcrV-GST did not. Unlike YopR-GST, which can block type III machines only during their assembly, expression of YscP-GST led to an immediate and complete block of all secretion. The secretion signal of YscP was mapped to its first 10 codons or amino acids; however, YscP(Delta 2-15)-GST, lacking this secretion signal, imposed a partial blockade. YscP-GST copurified with the type III ATPase complex (YscN, YscL, and YscQ) and with YscO, suggesting that the association of specific machine components with the impassable substrate may cause the block in type III secretion.

Novel Screen to Assess Bactericidal Activity of Compounds Against Non-replicating Mycobacterium abscessus.

Mycobacterium abscessus infections are increasing worldwide. Current drug regimens are largely ineffective, yet the current development pipeline for M. abscessus is alarmingly sparse. Traditional discovery efforts for M. abscessus assess the capability of a new drug to inhibit bacterial growth under nutrient-rich growth conditions, but this does not predict the impact when used in the clinic. The disconnect between in vitro and in vivo activity is likely due to the genetic and physiological adaptation of the bacteria to the environmental conditions encountered during infection; these include low oxygen tension and nutrient starvation. We sought to fill a gap in the drug discovery pipeline by establishing an assay to identify novel compounds with bactericidal activity against M. abscessus under non-replicating conditions. We developed and validated a novel screen using nutrient starvation to generate a non-replicating state. We used alamarBlue® to measure metabolic activity and demonstrated this correlates with bacterial viability under these conditions. We optimized key parameters and demonstrated reproducibility. Using this assay, we determined that niclosamide was bactericidal against non-replicating bacilli, highlighting its potential to be included in M. abscessus regimens. In contrast, most other drugs currently used in the clinic for M. abscessus infections, were completely inactive, potentially explaining their poor efficacy. Thus, our assay allows for rapid identification of bactericidal compounds in a model using conditions that are more relevant in vivo. This screen can be used in a high-throughput way to identify novel agents with properties that promise an increase in efficacy, while also shortening treatment times.

Improved Phenoxyalkylbenzimidazoles with Activity against Mycobacterium tuberculosis Appear to Target QcrB.

The phenoxy alkyl benzimidazoles (PABs) have good antitubercular activity. We expanded our structure-activity relationship studies to determine the core components of PABs required for activity. The most potent compounds had minimum inhibitory concentrations against Mycobacterium tuberculosis in the low nanomolar range with very little cytotoxicity against eukaryotic cells as well as activity against intracellular bacteria. We isolated resistant mutants against PAB compounds, which had mutations in either Rv1339, of unknown function, or qcrB, a component of the cytochrome bc1 oxidase of the electron transport chain. QcrB mutant strains were resistant to all PAB compounds, whereas Rv1339 mutant strains were only resistant to a subset, suggesting that QcrB is the target. The discovery of the target for PAB compounds will allow for the improved design of novel compounds to target intracellular M. tuberculosis.