T-SPOT.TB Reactivity in Southern African Children With and Without in Utero Human Immunodeficiency Virus Exposure.

Infants who are human immunodeficiency virus (HIV)-exposed uninfected (iHEU) experience higher risk of infectious morbidity than infants HIV-unexposed uninfected (iHUU). We compared tuberculosis (TB) infection prevalence in 418 Bacillus Calmette-Guérin vaccinated sub-Saharan African iHEU and iHUU aged 9-18 months using T-SPOT.TB. Prevalence of TB infection was low and did not differ by HIV exposure status.

Nanomaterials-based vaccines to target intracellular bacterial pathogens.

Development of novel immunization approaches to combat a growing list of emerging and ancient infectious agents is a global health priority. Intensive efforts over the last several decades have identified alternative approaches to improve upon traditional vaccines that are based on live, attenuated agents, or formulations of inactivated agents with adjuvants. Rapid advances in RNA-based and other delivery systems for immunization have recently revolutionized the potential to protect populations from viral pathogens, such as SARS-CoV-2. Similar efforts to combat bacterial pathogens, especially species with an intracellular niche, have lagged significantly. In the past decade, advances in nanotechnology have yielded a variety of new antigen/adjuvant carrier systems for use in vaccine development against infectious viruses and bacteria. The tunable properties of nanomaterial-based vaccines allow for balancing immunogenicity and safety which is a key hurdle in traditional antigen and adjuvant formulations. In this review, we discuss several novel nanoparticle-based vaccine platforms that show promise for use against intracellular bacteria as demonstrated by the feasibility of construction, enhanced antigen presentation, induction of cell mediated and humoral immune responses, and improved survival outcomes in in vivo models.

Novel chemical entities inhibiting Mycobacterium tuberculosis growth identified by phenotypic high-throughput screening.

We performed a high-throughput phenotypic whole cell screen of Mycobacterium tuberculosis against a diverse chemical library of approximately 100,000 compounds from the AbbVie corporate collection and identified 24 chemotypes with anti-tubercular activity. We selected two series for further exploration and conducted structure-activity relationship studies with new analogs for the 4-phenyl piperidines (4PP) and phenylcyclobutane carboxamides (PCB). Strains with mutations in MmpL3 demonstrated resistance to both compound series. We isolated resistant mutants for the two series and found mutations in MmpL3. These data suggest that MmpL3 is the target, or mechanism of resistance for both series.

Self-adjuvanting nanovaccines boost lung-resident CD4+ T cell immune responses in BCG-primed mice.

Heterologous vaccine regimens could extend waning protection in the global population immunized with Mycobacterium bovis Bacille Calmette-Guerin (BCG). We demonstrate that pulmonary delivery of peptide nanofibers (PNFs) bearing an Ag85B CD4+ T cell epitope increased the frequency of antigen-specific T cells in BCG-primed mice, including heterogenous populations with tissue resident memory (Trm) and effector memory (Tem) phenotype, and functional cytokine recall. Adoptive transfer of dendritic cells pulsed with Ag85B-bearing PNFs further expanded the frequency and functional repertoire of memory CD4+ T cells. Transcriptomic analysis suggested that the adjuvanticity of peptide nanofibers is, in part, due to the release of damage-associated molecular patterns. A single boost with monovalent Ag85B PNF in BCG-primed mice did not reduce lung bacterial burden compared to BCG alone following aerosol Mtb challenge. These findings support the need for novel BCG booster strategies that activate pools of Trm cells with potentially diverse localization, trafficking, and immune function.

Baseline mapping of Oropouche virology, epidemiology, therapeutics, and vaccine research and development.

Oropouche virus (OROV) is an arthropod-borne orthobunyavirus found in South America and causes Oropouche fever, a febrile infection similar to dengue. It is the second most prevalent arthropod-borne viral disease in South America after dengue. Over 500,000 cases have been diagnosed since the virus was first discovered in 1955; however, this is likely a significant underestimate given the limited availability of diagnostics. No fatalities have been reported to date, however, up to 60% of cases have a recurrent phase of disease within one month of recovery from the primary disease course. The main arthropod vector is the biting midge Culicoides paraensis, which has a geographic range as far north as the United States and demonstrates the potential for OROV to geographically expand. The transmission cycle is incompletely understood and vertebrate hosts include both non-human primates and birds further supporting the potential ability of the virus to spread. A number of candidate antivirals have been evaluated against OROV in vitro but none showed antiviral activity. Surprisingly, there is only one report in the literature on candidate vaccines. We suggest that OROV is an undervalued pathogen much like chikungunya, Schmallenberg, and Zika viruses were before they emerged. Overall, OROV is an important emerging disease that has been under-investigated and has the potential to cause large epidemics in the future. Further research, in particular candidate vaccines, is needed for this important pathogen.

Coiled Coil Crosslinked Alginate Hydrogels Dampen Macrophage-Driven Inflammation.

Alginate hydrogels are widely used for tissue engineering and regenerative medicine due to their excellent biocompatibility. A facile and commonly used strategy to crosslink alginate is the addition of Ca2+ that leads to hydrogelation. However, extracellular Ca2+ is a secondary messenger in activating inflammasome pathways following physical injury or pathogenic insult, which carries the risk of persistent inflammation and scaffold rejection. Here, we present graft copolymers of charge complementary heterodimeric coiled coil (CC) peptides and alginate that undergo supramolecular self-assembly to form Ca2+ free alginate hydrogels. The formation of heterodimeric CCs was confirmed using circular dichroism spectroscopy, and scanning electron microscopy revealed a significant difference in crosslink density and homogeneity between Ca2+ and CC crosslinked gels. The resulting hydrogels were self-supporting and display shear-thinning and shear-recovery properties. In response to lipopolysaccharide (LPS) stimulation, peritoneal macrophages and bone marrow-derived dendritic cells cultured in the CC crosslinked gels exhibited a 10-fold reduction in secretion of the proinflammatory cytokine IL-1ß compared to Ca2+ crosslinked gels. A similar response was also observed in vivo upon peritoneal delivery of Ca2+ or CC crosslinked gels. Analysis of peritoneal lavage showed that macrophages in mice injected with Ca2+ crosslinked gels display a more inflammatory phenotype compared to macrophages from mice injected with CC crosslinked gels. These results suggest that CC peptides by virtue of their tunable sequence-structure-function relationship and mild gelation conditions are promising alternative crosslinkers for alginate and other biopolymer scaffolds used in tissue engineering.

Peptide-based supramolecular vaccine systems.

Currently approved replication-competent and inactivated vaccines are limited by excessive reactogenicity and poor safety profiles, while subunit vaccines are often insufficiently immunogenic without co-administering exogenous adjuvants. Self-assembling peptide-, peptidomimetic-, and protein-based biomaterials offer a means to overcome these challenges through their inherent modularity, multivalency, and biocompatibility. As these scaffolds are biologically derived and present antigenic arrays reminiscent of natural viruses, they are prone to immune recognition and are uniquely capable of functioning as self-adjuvanting vaccine delivery vehicles that improve humoral and cellular responses. Beyond this intrinsic immunological advantage, the wide range of available amino acids allows for facile de novo design or straightforward modifications to existing sequences. This has permitted the development of vaccines and immunotherapies tailored to specific disease models, as well as generalizable platforms that have been successfully applied to prevent or treat numerous infectious and non-infectious diseases. In this review, we briefly introduce the immune system, discuss the structural determinants of coiled coils, ß-sheets, peptide amphiphiles, and protein subunit nanoparticles, and highlight the utility of these materials using notable examples of their innate and adaptive immunomodulatory capacity. STATEMENT OF SIGNIFICANCE: Subunit vaccines have recently gained considerable attention due to their favorable safety profiles relative to traditional whole-cell vaccines; however, their reduced efficacy requires co-administration of reactogenic adjuvants to boost immune responses. This has led to collaborative efforts between engineers and immunologists to develop nanomaterial-based vaccination platforms that can elicit protection without deleterious side effects. Self-assembling peptidic biomaterials are a particularly attractive approach to this problem, as their structure and function can be controlled through primary sequence design and their capacity for multivalent presentation of antigens grants them intrinsic self-adjuvanticity. This review introduces the various architectures adopted by self-assembling peptides and discusses their application as modulators of innate and adaptive immunity.

Dual activity of niclosamide to suppress replication of integrated HIV-1 and Mycobacterium tuberculosis (Beijing).

The human immunodeficiency virus (HIV) pandemic is driving the re-emergence of tuberculosis (TB) as a global health threat, both by increasing the susceptibility of HIV-infected people to infection with Mycobacterium tuberculosis (Mtb), and increasing the rate of emergence of drug-resistant Mtb. There are several other clinical challenges for treatment of co-infected patients including: expense, pill burden, toxicity, and malabsorption that further necessitate the search for new drugs that may be effective against both pathogens simultaneously. The anti-helminthic niclosamide has been shown to have activity against a laboratory strain of Mtb in liquid culture while bacteriostatic activity against non-replicating M. abscessus was also recently described. Here we extend these findings to further demonstrate that niclosamide inhibits mycobacterial growth in infected human macrophages and mediates potent bacteriostatic activity against the virulent Mtb Beijing strain. Importantly, we provide the first evidence that niclosamide inhibits HIV replication in human macrophages and Jurkat T cells through post-integration effects on pro-virus transcription. The dual antiviral and anti-mycobacterial activity was further observed in an in vitro model of HIV and Mtb co-infection using human primary monocyte-derived macrophages. These results support further investigation of niclosamide and derivatives as anti-retroviral/anti-mycobacterial agents that may reduce clinical challenges associated with multi-drug regimens and drug resistance.

8-Hydroxyquinolines are bactericidal against Mycobacterium tuberculosis.

There is an urgent need for new treatments effective against Mycobacterium tuberculosis, the causative agent of tuberculosis. The 8-hydroxyquinoline series is a privileged scaffold with anticancer, antifungal, and antibacterial activities. We conducted a structure-activity relationship study of the series regarding its antitubercular activity using 26 analogs. The 8-hydroxyquinolines showed good activity against M. tuberculosis, with minimum inhibitory concentrations (MIC90) of <5 µM for some analogs. Small substitutions at C5 resulted in the most potent activity. Substitutions at C2 generally decreased potency, although a sub-family of 2-styryl-substituted analogs retained activity. Representative compounds demonstrated bactericidal activity against replicating M. tuberculosis with >4 log kill at 10× MIC over 14 days. The majority of the compounds demonstrated cytotoxicity (IC50 of <100 µM). Further development of this series as antitubercular agents should address the cytotoxicity liability. However, the 8-hydroxyquinoline series represents a useful tool for chemical genomics to identify novel targets in M. tuberculosis.

A potent Lassa virus antiviral targets an arenavirus virulence determinant.

Arenaviruses are a significant cause of hemorrhagic fever, an often-fatal disease for which there is no approved antiviral therapy. Lassa fever in particular generates high morbidity and mortality in West Africa, where the disease is endemic, and a recent outbreak in Nigeria was larger and more geographically diverse than usual. We are developing LHF-535, a small-molecule viral entry inhibitor that targets the arenavirus envelope glycoprotein, as a therapeutic candidate for Lassa fever and other hemorrhagic fevers of arenavirus origin. Using a lentiviral pseudotype infectivity assay, we determined that LHF-535 had sub-nanomolar potency against the viral envelope glycoproteins from all Lassa virus lineages, with the exception of the glycoprotein from the LP strain from lineage I, which was 100-fold less sensitive than that of other strains. This reduced sensitivity was mediated by a unique amino acid substitution, V434I, in the transmembrane domain of the envelope glycoprotein GP2 subunit. This position corresponds to the attenuation determinant of Candid#1, a live-attenuated Junín virus vaccine strain used to prevent Argentine hemorrhagic fever. Using a virus-yield reduction assay, we determined that LHF-535 potently inhibited Junín virus, but not Candid#1, and the Candid#1 attenuation determinant, F427I, regulated this difference in sensitivity. We also demonstrated that a daily oral dose of LHF-535 at 10 mg/kg protected mice from a lethal dose of Tacaribe virus. Serial passage of Tacaribe virus in LHF-535-treated Vero cells yielded viruses that were resistant to LHF-535, and the majority of drug-resistant viruses exhibited attenuated pathogenesis. These findings provide a framework for the clinical development of LHF-535 as a broad-spectrum inhibitor of arenavirus entry and provide an important context for monitoring the emergence of drug-resistant viruses.

A class of hydrazones are active against non-replicating Mycobacterium tuberculosis.

There is an urgent need for the development of shorter, simpler and more tolerable drugs to treat antibiotic tolerant populations of Mycobacterium tuberculosis. We previously identified a series of hydrazones active against M. tuberculosis. We selected five representative compounds for further analysis. All compounds were active against non-replicating M. tuberculosis, with two compounds demonstrating greater activity under hypoxic conditions than aerobic culture. Compounds had bactericidal activity with MBC/MIC of < 4 and demonstrated an inoculum-dependent effect against aerobically replicating bacteria. Bacterial kill kinetics demonstrated a faster rate of kill against non-replicating bacilli generated by nutrient starvation. Compounds had limited activity against other bacterial species. In conclusion, we have demonstrated that hydrazones have some attractive properties in terms of their anti-tubercular activity.

Construction of an overexpression library for Mycobacterium tuberculosis.

There is a pressing need to develop novel anti-tubercular drugs. High-throughput phenotypic screening yields chemical series that inhibit bacterial growth. Target identification for such series is challenging, but necessary for optimization of target engagement and the development of series into clinical drugs. We constructed a library of recombinant Mycobacterium tuberculosis strains each expressing a single protein from an inducible promoter as a tool for target identification. The library of 1733 clones was arrayed in 96-well plates for rapid screening and monitoring growth. The library contains the majority of the annotated essential genes as well as genes involved in cell wall and fatty acid biosynthesis, virulence factors, regulatory proteins, efflux, and respiration pathways. We evaluated the growth kinetics and plasmid stability over three passages for each clone in the library. We determined expression levels (mRNA and/or protein) in 396 selected clones. We screened the entire library and identified the Alr-expressing clone as the only recombinant strain, which grew in the presence of d-cycloserine (DCS). We confirmed that the Alr-expressing clone was resistant to DCS (7-fold shift in minimum inhibitory concentration). The library represents a new tool that can be used to screen for compound resistance and other phenotypes.

In Vitro Evaluation of Novel Nitazoxanide Derivatives against Mycobacterium tuberculosis.

Nitazoxanide has antiparasitic and antibiotic activities including activity against Mycobacterium tuberculosis. We prepared and evaluated a set of its analogues to determine the structure-activity relationship, and identified several amide- and urea-based analogues with low micromolar activity against M. tuberculosis in vitro. Pharmacokinetics in the rat suggested a path forward to obtain bioavailable compounds. The series had a good microbiological profile with bactericidal activity in vitro against replicating and nonreplicating M. tuberculosis. Analogues had limited activity against other Gram-positive bacteria but no activity against Gram-negative bacteria. Our studies identified the key liability in this series as cytotoxicity. Future work concentrating on identifying the target(s) could assist in removing activity against eukaryotic cells.

A Target-Based Whole Cell Screen Approach To Identify Potential Inhibitors of Mycobacterium tuberculosis Signal Peptidase.

The general secretion (Sec) pathway is a conserved essential pathway in bacteria and is the primary route of protein export across the cytoplasmic membrane. During protein export, the signal peptidase LepB catalyzes the cleavage of the signal peptide and subsequent release of mature proteins into the extracellular space. We developed a target-based whole cell assay to screen for potential inhibitors of LepB, the sole signal peptidase in Mycobacterium tuberculosis, using a strain engineered to underexpress LepB (LepB-UE). We screened 72,000 compounds against both the Lep-UE and wild-type (wt) strains. We identified the phenylhydrazone (PHY) series as having higher activity against the LepB-UE strain. We conducted a limited structure-activity relationship determination around a representative PHY compound with differential activity (MICs of 3.0 µM against the LepB-UE strain and 18 µM against the wt); several analogues were less potent against the LepB overexpressing strain. A number of chemical modifications around the hydrazone moiety resulted in improved potency. Inhibition of LepB activity was observed for a number of compounds in a biochemical assay using cell membrane fraction derived from M. tuberculosis. Compounds did not increase cell permeability, dissipate membrane potential, or inhibit an unrelated mycobacterial enzyme, suggesting a specific mode of action related to the LepB secretory mechanism.

Oxadiazoles Have Butyrate-Specific Conditional Activity against Mycobacterium tuberculosis.

Mycobacterium tuberculosis is a global pathogen of huge importance which can adapt to several host niche environments in which carbon source availability is likely to vary. We developed and ran a phenotypic screen using butyrate as the sole carbon source to be more reflective of the host lung environment. We screened a library of ∼87,000 small compounds and identified compounds which demonstrated good antitubercular activity against M. tuberculosis grown with butyrate but not with glucose as the carbon source. Among the hits, we identified an oxadiazole series (six compounds) which had specific activity against M. tuberculosis but which lacked cytotoxicity against mammalian cells.

Optimization and Evaluation of 5-Styryl-Oxathiazol-2-one Mycobacterium tuberculosis Proteasome Inhibitors as Potential Antitubercular Agents.

This is the first report of 5-styryl-oxathiazol-2-ones as inhibitors of the Mycobacterium tuberculosis (Mtb) proteasome. As part of the study, the structure-activity relationship of oxathiazolones as Mtb proteasome inhibitors has been investigated. Furthermore, the prepared compounds displayed a good selectivity profile for Mtb compared to the human proteasome. The 5-styryl-oxathiazol-2-one inhibitors identified showed little activity against replicating Mtb, but were rapidly bactericidal against nonreplicating bacteria. (E)-5-(4-Chlorostyryl)-1,3,4-oxathiazol-2-one) was most effective, reducing the colony-forming units (CFU)/mL below the detection limit in only seven days at all concentrations tested. The results suggest that this new class of Mtb proteasome inhibitors has the potential to be further developed into novel antitubercular agents for synergistic combination therapies with existing drugs.

Identification of Phenoxyalkylbenzimidazoles with Antitubercular Activity.

We conducted an evaluation of the phenoxyalkylbenzimidazole series based on the exemplar 2-ethyl-1-(3-phenoxypropyl)-1H-benzo[d]imidazole for its antitubercular activity. Four segments of the molecule were examined systematically to define a structure-activity relationship with respect to biological activity. Compounds had submicromolar activity against Mycobacterium tuberculosis; the most potent compound had a minimum inhibitory concentration (MIC) of 52 nM and was not cytotoxic against eukaryotic cells (selectivity index = 523). Compounds were selective for M. tuberculosis over other bacterial species, including the closely related Mycobacterium smegmatis. Compounds had a bacteriostatic effect against aerobically grown, replicating M. tuberculosis, but were bactericidal against nonreplicating bacteria. Representative compounds had moderate to high permeability in MDCK cells, but were rapidly metabolized in rodents and human liver microsomes, suggesting the possibility of rapid in vivo hepatic clearance mediated by oxidative metabolism. These results indicate that the readily synthesized phenoxyalkylbenzimidazoles are a promising class of potent and selective antitubercular agents, if the metabolic liability can be solved.

Synthesis and anti-tubercular activity of 3-substituted benzo[b]thiophene-1,1-dioxides.

We demonstrated that the 3-substituted benzothiophene-1,1-dioxide class of compounds are effective inhibitors of Mycobacterium tuberculosis growth under aerobic conditions. We examined substitution at the C-3 position of the benzothiophene-1,1-dioxide series systematically to delineate structure-activity relationships influencing potency and cytotoxicity. Compounds were tested for inhibitory activity against virulent M. tuberculosis and eukaryotic cells. The tetrazole substituent was most potent, with a minimum inhibitory concentration (MIC) of 2.6 µM. However, cytotoxicity was noted with even more potency (Vero cell TC50 = 0.1 µM). Oxadiazoles had good anti-tubercular activity (MICs of 3-8 µM), but imidazoles, thiadiazoles and thiazoles had little activity. Cytotoxicity did not track with anti-tubercular activity, suggesting different targets or mode of action between bacterial and eukaryotic cells. However, we were unable to derive analogs without cytotoxicity; all compounds synthesized were cytotoxic (TC50 of 0.1-5 µM). We conclude that cytotoxicity is a liability in this series precluding it from further development. However, the series has potent anti-tubercular activity and future efforts towards identifying the mode of action could result in the identification of novel drug targets.

Identification of the translational start site of codon-optimized mCherry in Mycobacterium tuberculosis.

BACKGROUND: Fluorescent proteins are used widely as reporter genes in many organisms. We previously codon-optimized mCherry for Mycobacterium tuberculosis and generated expression constructs with high level expression in mycobacteria with multiple uses in vitro and in vivo. However, little is known about the expression of fluorescent proteins in mycobacteria and the translational start codon for mCherry has not been experimentally determined. RESULTS: We determined the translational start site for functional (fluorescent) mCherry in mycobacteria. Several potential translational start codons were identified; introduction of downstream stop codons by mutagenesis was used to determine which start codon was utilized in the bacterial cells. Fluorescent protein was expressed from a construct which would allow translation of a protein of 226 amino acids or a protein of 235 amino acids. No fluorescence was seen when a construct which could give rise to a protein of 219 amino acids was used. Similar results were obtained in mycobacteria and in Escherichia coli. Western blotting confirmed that mCherry was expressed from the constructs encoding 235 or 226 amino acids, but not from the plasmid encoding 219 amino acids. N-terminal sequencing and mass determination confirmed that the mature protein was 226 amino acids and commenced with the amino acid sequence AIIKE. CONCLUSION: We conclude that mCherry is expressed in M. tuberculosis as a smaller protein than expected lacking the GFP-derived N-terminal sequence designed to allow efficient fusions.