Immunizations with diverse sarbecovirus receptor-binding domains elicit SARS-CoV-2 neutralizing antibodies against a conserved site of vulnerability.

Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Second-generation vaccines will need to elicit neutralizing antibodies against sites that are evolutionarily conserved across the sarbecovirus subgenus. Here, we immunized mice containing a human antibody repertoire with diverse sarbecovirus receptor-binding domains (RBDs) to identify antibodies targeting conserved sites of vulnerability. Antibodies with broad reactivity against diverse clade B RBDs targeting the conserved class 4 epitope, with recurring IGHV/IGKV pairs, were readily elicited but were non-neutralizing. However, rare class 4 antibodies binding this conserved RBD supersite showed potent neutralization of SARS-CoV-2 and all variants of concern. Structural analysis revealed that the neutralizing ability of cross-reactive antibodies was reserved only for those with an elongated CDRH3 that extends the antiparallel beta-sheet RBD core and orients the antibody light chain to obstruct ACE2-RBD interactions. These results identify a structurally defined pathway for vaccine strategies eliciting escape-resistant SARS-CoV-2 neutralizing antibodies.

A Liver Capsular Network of Monocyte-Derived Macrophages Restricts Hepatic Dissemination of Intraperitoneal Bacteria by Neutrophil Recruitment.

The liver is positioned at the interface between two routes traversed by pathogens in disseminating infection. Whereas blood-borne pathogens are efficiently cleared in hepatic sinusoids by Kupffer cells (KCs), it is unknown how the liver prevents dissemination of peritoneal pathogens accessing its outer membrane. We report here that the hepatic capsule harbors a contiguous cellular network of liver-resident macrophages phenotypically distinct from KCs. These liver capsular macrophages (LCMs) were replenished in the steady state from blood monocytes, unlike KCs that are embryonically derived and self-renewing. LCM numbers increased after weaning in a microbiota-dependent process. LCMs sensed peritoneal bacteria and promoted neutrophil recruitment to the capsule, and their specific ablation resulted in decreased neutrophil recruitment and increased intrahepatic bacterial burden. Thus, the liver contains two separate and non-overlapping niches occupied by distinct resident macrophage populations mediating immunosurveillance at these two pathogen entry points to the liver.

L-selectin-dependent and -independent homing of naïve lymphocytes through the lung draining lymph node support T cell response to pulmonary Mycobacterium tuberculosis infection.

Recruiting large numbers of naïve lymphocytes to lymph nodes is critical for mounting an effective adaptive immune response. While most naïve lymphocytes utilize homing molecule L-selectin to enter lymph nodes, some circulating cells can traffic to the lung-draining mediastinal lymph node (mLN) through lymphatics via the intermediate organ, lung. However, whether this alternative trafficking mechanism operates in infection and contributes to T cell priming are unknown. We report that in pulmonary Mycobacterium tuberculosis-infected mice, homing of circulating lymphocytes to the mLN is significantly less efficient than to non-draining lymph node. CD62L blockade only partially reduced the homing of naïve T lymphocytes, consistent with L-selectin-independent routing of naïve lymphocytes to the site. We further demonstrated that lymphatic vessels in infected mLN expanded significantly and inhibiting lymphangiogenesis with a vascular endothelial growth factor receptor 3 kinase inhibitor reduced the recruitment of intravenously injected naïve lymphocytes to the mLN. Finally, mycobacterium-specific T cells entering via the L-selectin-independent route were readily activated in the mLN. Our study suggests that both L-selectin-dependent and -independent pathways contribute to naïve lymphocyte entry into mLN during M. tuberculosis infection and the latter pathway may represent an important mechanism for orchestrating host defence in the lungs.

Dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) is a cellular receptor for delta inulin adjuvant.

Delta inulin, or Advax, is a polysaccharide vaccine adjuvant that significantly enhances vaccine-mediated immune responses against multiple pathogens and was recently licensed for use in the coronavirus disease 2019 (COVID-19) vaccine SpikoGen. Although Advax has proven effective as an immune adjuvant, its specific binding targets have not been characterized. In this report, we identify a cellular receptor for Advax recognition. In vitro uptake of Advax particles by macrophage cell lines was substantially greater than that of latex beads of comparable size, suggesting an active uptake mechanism by phagocytic cells. Using a lectin array, Advax particles were recognized by lectins specific for various carbohydrate structures including mannosyl, N-acetylgalactosamine and galactose moieties. Expression in nonphagocytic cells of dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), a C-type lectin receptor, resulted in enhanced uptake of fluorescent Advax particles compared with mock-transfected cells. Advax uptake was reduced with the addition of ethylenediaminetetraacetic acid and mannan to cells, which are known inhibitors of DC-SIGN function. Finally, a specific blockade of DC-SIGN using a neutralizing antibody abrogated Advax uptake in DC-SIGN-expressing cells. Together, these results identify DC-SIGN as a putative receptor for Advax. Given the known immunomodulatory role of DC-SIGN, the findings described here have implications for the use of Advax adjuvants in humans and inform future mechanistic studies.

Synthetic protein conjugate vaccines provide protection against Mycobacterium tuberculosis in mice.

The global incidence of tuberculosis remains unacceptably high, with new preventative strategies needed to reduce the burden of disease. We describe here a method for the generation of synthetic self-adjuvanted protein vaccines and demonstrate application in vaccination against Mycobacterium tuberculosis Two vaccine constructs were designed, consisting of full-length ESAT6 protein fused to the TLR2-targeting adjuvants Pam2Cys-SK4 or Pam3Cys-SK4 These were produced by chemical synthesis using a peptide ligation strategy. The synthetic self-adjuvanting vaccines generated powerful local CD4+ T cell responses against ESAT6 and provided significant protection in the lungs from virulent M. tuberculosis aerosol challenge when administered to the pulmonary mucosa of mice. The flexible synthetic platform we describe, which allows incorporation of adjuvants to multiantigenic vaccines, represents a general approach that can be applied to rapidly assess vaccination strategies in preclinical models for a range of diseases, including against novel pandemic pathogens such as SARS-CoV-2.

Assembly of Immunogenic Protein Particles toward Advanced Synthetic Vaccines.

Immunogenic carrier proteins such as the non-toxic diphtheria toxin variant, cross-reacting material 197 (CRM197), are widely used in subunit vaccine formulations to boost immunogenicity of chemically conjugated antigens. Conjugate vaccines are inherently expensive due to laborious manufacturing steps. Here, this work develops a particulate vaccine platform based on using engineered Escherichia coli to assemble CRM197-antigen fusion proteins into discrete submicron-sized particles. This approach enables precise loading of diverse antigens and epitopes enhancing their immunogenicity. A cost-effective, high-yield, and scalable biomanufacturing process is developed. Purified particulate CRM197-antigen vaccines are ambient-temperature stable. CRM197 particles incorporating pathogen-specific antigens or epitopes from SARS-CoV-2, Streptococcus pyogenes (group A), and Mycobacterium tuberculosis induced cell-mediated and humoral immune responses mediating protective immunity in respective animal models of infection. The CRM197 particle vaccine platform is versatile, enabling co-delivery of selected antigens/epitopes together with immunogenic CRM197 as discrete stable particles avoiding laborious manufacture of soluble CRM197 and antigen followed by chemical conjugation.

Mycobacterial infection-induced miR-206 inhibits protective neutrophil recruitment via the CXCL12/CXCR4 signalling axis.

Pathogenic mycobacteria actively dysregulate protective host immune signalling pathways during infection to drive the formation of permissive granuloma microenvironments. Dynamic regulation of host microRNA (miRNA) expression is a conserved feature of mycobacterial infections across host-pathogen pairings. Here we examine the role of miR-206 in the zebrafish model of Mycobacterium marinum infection, which allows investigation of the early stages of granuloma formation. We find miR-206 is upregulated following infection by pathogenic M. marinum and that antagomir-mediated knockdown of miR-206 is protective against infection. We observed striking upregulation of cxcl12a and cxcr4b in infected miR-206 knockdown zebrafish embryos and live imaging revealed enhanced recruitment of neutrophils to sites of infection. We used CRISPR/Cas9-mediated knockdown of cxcl12a and cxcr4b expression and AMD3100 inhibition of Cxcr4 to show that the enhanced neutrophil response and reduced bacterial burden caused by miR-206 knockdown was dependent on the Cxcl12/Cxcr4 signalling axis. Together, our data illustrate a pathway through which pathogenic mycobacteria induce host miR-206 expression to suppress Cxcl12/Cxcr4 signalling and prevent protective neutrophil recruitment to granulomas.

TCR Affinity Controls the Dynamics but Not the Functional Specification of the Antimycobacterial CD4+ T Cell Response.

The quality of T cell responses depends on the lymphocytes' ability to undergo clonal expansion, acquire effector functions, and traffic to the site of infection. Although TCR signal strength is thought to dominantly shape the T cell response, by using TCR transgenic CD4+ T cells with different peptide:MHC binding affinity, we reveal that TCR affinity does not control Th1 effector function acquisition or the functional output of individual effectors following mycobacterial infection in mice. Rather, TCR affinity calibrates the rate of cell division to synchronize the distinct processes of T cell proliferation, differentiation, and trafficking. By timing cell division-dependent IL-12R expression, TCR affinity controls when T cells become receptive to Th1-imprinting IL-12 signals, determining the emergence and magnitude of the Th1 effector pool. These findings reveal a distinct yet cooperative role for IL-12 and TCR binding affinity in Th1 differentiation and suggest that the temporal activation of clones with different TCR affinity is a major strategy to coordinate immune surveillance against persistent pathogens.

Bacillus Calmette-Guérin Skin Reaction Predicts Enhanced Mycobacteria-Specific T-Cell Responses in Infants: A Post Hoc Analysis of a Randomized Controlled Trial.

Rationale: Scar formation following bacillus Calmette-Guérin (BCG) vaccination has been associated with lower all-cause mortality; the relation between scar and mycobacteria-specific protection against tuberculosis is debated. Objectives: To evaluate the association between BCG skin reaction and mycobacteria-specific immune responses. Methods: A post hoc analysis was done among 214 infants in Australia randomized to vaccination with one of three BCG vaccine strains (BCG-Denmark, BCG-Japan, or BCG-Russia) given at birth or BCG-Denmark given at 2 months of age. Measurements and Main Results: BCG skin reaction size and characteristics 10 weeks after vaccination were related to the in vitro mycobacteria-specific immune responses measured in stimulated whole blood. The size and characteristics of the skin reaction correlated positively with in vitro immune responses, even after adjusting for BCG vaccine strain and age at vaccination. Specifically, the reaction size and characteristics correlated with the proportion of mycobacteria-specific polyfunctional CD4+ T cells after stimulation with BCG and PPD and, to a lesser extent, after stimulation with Mycobacterium tuberculosis or Mycobacterium ulcerans. A similar correlation was observed with concentrations of IFN-γ, IL-2, tumor necrosis factor, and IL-13 in the supernatant after stimulation with BCG, PPD, and M. tuberculosis and to some degree for the proportions of mycobacteria-specific polyfunctional CD8+ T cells and CD107+ cytotoxic cells. Conclusions: BCG skin reaction correlated with the magnitude of mycobacteria-specific T-cell responses. As T-cell responses play a key role in defense against mycobacteria, the relationship between BCG scar formation and protection against tuberculosis should be revisited. This may also extend to the need for BCG revaccination in scar-negative individuals.Clinical trial registered with www.australianclinicaltrials.gov.au/clinical-trial-registries (ACTRN12608000227392).

Increased SARS-CoV-2 Infection, Protease, and Inflammatory Responses in Chronic Obstructive Pulmonary Disease Primary Bronchial Epithelial Cells Defined with Single-Cell RNA Sequencing.

Rationale: Patients with chronic obstructive pulmonary disease (COPD) develop more severe coronavirus disease (COVID-19); however, it is unclear whether they are more susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and what mechanisms are responsible for severe disease. Objectives: To determine whether SARS-CoV-2 inoculated primary bronchial epithelial cells (pBECs) from patients with COPD support greater infection and elucidate the effects and mechanisms involved. Methods: We performed single-cell RNA sequencing analysis on differentiated pBECs from healthy subjects and patients with COPD 7 days after SARS-CoV-2 inoculation. We correlated changes with viral titers, proinflammatory responses, and IFN production. Measurements and Main Results: Single-cell RNA sequencing revealed that COPD pBECs had 24-fold greater infection than healthy cells, which was supported by plaque assays. Club/goblet and basal cells were the predominant populations infected and expressed mRNAs involved in viral replication. Proteases involved in SARS-CoV-2 entry/infection (TMPRSS2 and CTSB) were increased, and protease inhibitors (serpins) were downregulated more so in COPD. Inflammatory cytokines linked to COPD exacerbations and severe COVID-19 were increased, whereas IFN responses were blunted. Coexpression analysis revealed a prominent population of club/goblet cells with high type 1/2 IFN responses that were important drivers of immune responses to infection in both healthy and COPD pBECs. Therapeutic inhibition of proteases and inflammatory imbalances reduced viral titers and cytokine responses, particularly in COPD pBECs. Conclusions: COPD pBECs are more susceptible to SARS-CoV-2 infection because of increases in coreceptor expression and protease imbalances and have greater inflammatory responses. A prominent cluster of IFN-responsive club/goblet cells emerges during infection, which may be important drivers of immunity. Therapeutic interventions suppress SARS-CoV-2 replication and consequent inflammation.

Patient and Clinician Perceptions of the Pulse Oximeter in a Remote Monitoring Setting for COVID-19: Qualitative Study.

BACKGROUND: As a response to the COVID-19 pandemic, the Sydney Local Health District in New South Wales, Australia, launched the rpavirtual program, the first full-scale virtual hospital in Australia, to remotely monitor and follow up stable patients with COVID-19. As part of the intervention, a pulse oximeter wearable device was delivered to patients to monitor their oxygen saturation levels, a critical indicator of COVID-19 patient deterioration. Understanding users' perceptions toward the device is fundamental to assessing its usability and acceptability and contributing to the effectiveness of the intervention, but no research to date has explored the user experience of the pulse oximeter for remote monitoring in this setting. OBJECTIVE: This study aimed to explore the use, performance, and acceptability of the pulse oximeter by clinicians and patients in rpavirtual during COVID-19. METHODS: Semistructured interviews and usability testing were conducted. Stable adult patients with COVID-19 (aged ≥18 years) who used the pulse oximeter and were monitored by rpavirtual, and rpavirtual clinicians monitoring these patients were interviewed. Clinicians could be nurses, doctors, or staff who were part of the team that assisted patients with the use of the pulse oximeter. Usability testing was conducted with patients who had the pulse oximeter when they were contacted. Interviews were coded using the Theoretical Framework of Acceptability. Usability testing was conducted using a think-aloud protocol. Data were collected until saturation was reached. RESULTS: Twenty-one patients (average age 51, SD 13 years) and 15 clinicians (average age 41, SD 11 years) completed the interview. Eight patients (average age 51, SD 13 years) completed the usability testing. All participants liked the device and thought it was easy to use. They also had a good understanding of how to use the device and the device's purpose. Patients' age and device use-related characteristics (eg, the warmth of hands and hand steadiness) were identified by users as factors negatively impacting the accurate use of the pulse oximeter. CONCLUSIONS: Patients and clinicians had very positive perceptions of the pulse oximeter for COVID-19 remote monitoring, indicating high acceptability and usability of the device. However, factors that may impact the accuracy of the device should be considered when delivering interventions using the pulse oximeter for remote monitoring. Targeted instructions about the use of the device may be necessary for specific populations (eg, older people and patients unfamiliar with technology). Further research should focus on the integration of the pulse oximeter data into electronic medical records for real-time and secure patient monitoring.

Community-wide Screening for Tuberculosis in a High-Prevalence Setting.

BACKGROUND: The World Health Organization has set ambitious targets for the global elimination of tuberculosis. However, these targets will not be achieved at the current rate of progress. METHODS: We performed a cluster-randomized, controlled trial in Ca Mau Province, Vietnam, to evaluate the effectiveness of active community-wide screening, as compared with standard passive case detection alone, for reducing the prevalence of tuberculosis. Persons 15 years of age or older who resided in 60 intervention clusters (subcommunes) were screened for pulmonary tuberculosis, regardless of symptoms, annually for 3 years, beginning in 2014, by means of rapid nucleic acid amplification testing of spontaneously expectorated sputum samples. Active screening was not performed in the 60 control clusters in the first 3 years. The primary outcome, measured in the fourth year, was the prevalence of microbiologically confirmed pulmonary tuberculosis among persons 15 years of age or older. The secondary outcome was the prevalence of tuberculosis infection, as assessed by an interferon gamma release assay in the fourth year, among children born in 2012. RESULTS: In the fourth-year prevalence survey, we tested 42,150 participants in the intervention group and 41,680 participants in the control group. A total of 53 participants in the intervention group (126 per 100,000 population) and 94 participants in the control group (226 per 100,000) had pulmonary tuberculosis, as confirmed by a positive nucleic acid amplification test for Mycobacterium tuberculosis (prevalence ratio, 0.56; 95% confidence interval [CI], 0.40 to 0.78; P<0.001). The prevalence of tuberculosis infection in children born in 2012 was 3.3% in the intervention group and 2.6% in the control group (prevalence ratio, 1.29; 95% CI, 0.70 to 2.36; P = 0.42). CONCLUSIONS: Three years of community-wide screening in persons 15 years of age or older who resided in Ca Mau Province, Vietnam, resulted in a lower prevalence of pulmonary tuberculosis in the fourth year than standard passive case detection alone. (Funded by the Australian National Health and Medical Research Council; ACT3 Australian New Zealand Clinical Trials Registry number, ACTRN12614000372684.).

Treatment of infection-induced vascular pathologies is protective against persistent rough morphotype Mycobacterium abscessus infection in zebrafish.

Mycobacterium abscessus infections are of increasing global prevalence and are often difficult to treat due to complex antibiotic resistance profiles. While there are similarities between the pathogenesis of M. abscessus and tuberculous mycobacteria, including granuloma formation and stromal remodelling, there are distinct molecular differences at the host-pathogen interface. Here we have used a zebrafish-M. abscessus model and host-directed therapies that were previously identified in the zebrafish-M. marinum model to identify potential host-directed therapies against M. abscessus infection. We find efficacy of anti-angiogenic and vascular normalizing therapies against rough M. abscessus infection, but no effect of anti-platelet drugs.

Biological and Biochemical Evaluation of Isatin-Isoniazid Hybrids as Bactericidal Candidates against Mycobacterium tuberculosis.

Tuberculosis remains a leading cause of mortality among infectious diseases worldwide, prompting the need to discover new drugs to fight this disease. We report here the design, synthesis, and antimycobacterial activity of isatin-mono/bis-isoniazid hybrids. Most of the compounds exhibited very high activity against Mycobacterium tuberculosis, with MICs in the range of 0.195 to 0.39 µg/ml, and exerted a more potent bactericidal effect than the standard antitubercular drug isoniazid (INH). Importantly, these compounds were found to be well tolerated at high doses (>200 µg/ml) on Vero kidney cells, leading to high selectivity indices. Two of the most promising hybrids were evaluated for activity in THP-1 macrophages infected with M. tuberculosis, among which compound 11e was found to be slightly more effective than INH. Overexpression of InhA along with cross-resistance determination of the most potent compounds, selection of resistant mutants, and biochemical analysis, allowed us to decipher their mode of action. These compounds effectively inhibited mycolic acid biosynthesis and required KatG to exert their biological effects. Collectively, this suggests that the synthesized isatin-INH hybrids are promising antitubercular molecules for further evaluation in preclinical settings.

Household-Contact Investigation for Detection of Tuberculosis in Vietnam.

BACKGROUND: Active case finding is a top priority for the global control of tuberculosis, but robust evidence for its effectiveness in high-prevalence settings is lacking. We sought to evaluate the effectiveness of household-contact investigation, as compared with standard, passive measures alone, in Vietnam. METHODS: We performed a cluster-randomized, controlled trial at clinics in 70 districts (local government areas with an average population of approximately 500,000 in urban areas and 100,000 in rural areas) in eight provinces of Vietnam. Health workers at each district clinic or hospital were assigned to perform either household-contact intervention plus standard passive case finding (intervention group) or passive case finding alone (control group). In the intervention districts, household contacts of patients with positive results for tuberculosis on sputum smear microscopy (smear-positive tuberculosis) were invited for clinical assessment and chest radiography at baseline and at 6, 12, and 24 months. The primary outcome was the cumulative incidence of registered cases of tuberculosis among household contacts of patients with tuberculosis during a 2-year period. RESULTS: In 70 selected districts, we enrolled 25,707 household contacts of 10,964 patients who had smear-positive pulmonary tuberculosis. In the 36 districts that were included in the intervention group, 180 of 10,069 contacts were registered as having tuberculosis (1788 cases per 100,000 population), as compared with 110 of 15,638 contacts (703 per 100,000) in the control group (relative risk of the primary outcome in the intervention group, 2.5; 95% confidence interval [CI], 2.0 to 3.2; P<0.001); the relative risk of smear-positive disease among household contacts in the intervention group was 6.4 (95% CI, 4.5 to 9.0; P<0.001). CONCLUSIONS: Household-contact investigation plus standard passive case finding was more effective than standard passive case finding alone for the detection of tuberculosis in a high-prevalence setting at 2 years. (Funded by the Australian National Health and Medical Research Council; ACT2 Australian New Zealand Clinical Trials Registry number, ACTRN12610000600044 .).

Macrophages of different tissue origin exhibit distinct inflammatory responses to mycobacterial infection.

Macrophages display marked plasticity with functions in both inflammation and tissue repair. Evidence demonstrates that this spectrum of macrophage phenotypes is influenced by their local microenvironment and tissue origin. However, in vitro macrophage experiments often do not or cannot readily use macrophages from the most relevant tissue of origin. This study investigated if the origin of two C57BL/6 mouse macrophage cell lines of alveolar (AMJ2-C11) and peritoneal (IC-21) origin may influence their response to mycobacterial infection. Both cell lines equally controlled the growth of Mycobacterium bovis BCG and Mycobacterium tuberculosis, although the expression of all proinflammatory cytokines and chemokines measured (TNF, IL-6, MCP-1, MIP-1α, MIP-1ß, and RANTES) was significantly higher in AMJ2-C11 cells than in IC-21 cells. During M. tuberculosis infection, IL-6, MCP-1, and RANTES expression increased 5-fold, and MIP-1ß expression increased 30-fold. Additionally, AMJ2-C11 cells exhibited significantly higher inducible nitric oxide synthase activity than IC-21 cells, indicative of a more polarized M1 response. The expression of multiple surface markers was also assessed by flow cytometry. CD80 and CD86 were significantly upregulated in AMJ2-C11 cells and downregulated in IC-21 cells during M. tuberculosis infection. The results support the notion that the origin of tissue-resident macrophages influences their phenotype and antimicrobial response and demonstrate hereto unrecognized potential for these cell lines in in vitro studies.

Tissue-resident regulatory T cells accumulate at human barrier lymphoid organs.

Regulatory T cells (Tregs) play a critical role in immune regulation and peripheral tolerance. While different types of Tregs have been identified in both mice and humans, much of our understanding about how these cells maintain immune homeostasis is derived from animal models. In this study, we examined two distinct human lymphoid organs to understand how repeated exposure to infections at the mucosal surface influences the phenotype and tissue localization of Tregs. We show that while Tregs in both tonsils and spleen express a tissue-resident phenotype, they accumulate in greater numbers in tonsils. Tonsillar-resident Tregs exhibit a highly suppressive phenotype with significantly increased expression of CD39, ICOS and CTLA-4 compared with their counterparts in circulation or in the spleen. Functionally, resident Tregs are able effectively to suppress T cell proliferation. We further demonstrate that tonsillar-resident Tregs share key features of T follicular helper cells. Spatial analysis reveals that the vast majority of resident Tregs are localized at the border of the T-zone and B cell follicle, as well as within the lymphocyte pockets enriched with resident memory T cells. Together our findings suggest that resident Tregs are strategically co-localized to maintain immune homeostasis at sites of recurrent inflammation.

Total Synthesis and Antimycobacterial Activity of Ohmyungsamycin†A, Deoxyecumicin, and Ecumicin.

The ohmyungsamycin and ecumicin natural product families are structurally related cyclic depsipeptides that display potent antimycobacterial activity. Herein the total syntheses of ohmyungsamycin A, deoxyecumicin, and ecumicin are reported, together with the direct biological comparison of members of these natural product families against Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB). The synthesis of each of the natural products employed a solid-phase strategy to assemble the linear peptide precursor, involving a key on-resin esterification and an optional on-resin dimethylation step, before a final solution-phase macrolactamization between the non-proteinogenic N-methyl-4-methoxy-l-tryptophan amino acid and a bulky N-methyl-l-valine residue. The synthetic natural products possessed potent antimycobacterial activity against Mtb with MIC90 's ranging from 110-360 nm and retained activity against Mtb in Mtb-infected macrophages. Deoxyecumicin also exhibited rapid bactericidal killing against Mtb, sterilizing cultures after 21 days.

Thrombocyte Inhibition Restores Protective Immunity to Mycobacterial Infection in Zebrafish.

BACKGROUND: Infection-induced thrombocytosis is a clinically important complication of tuberculosis infection. Recent studies have highlighted the utility of aspirin as a host-directed therapy modulating the inflammatory response to infection but have not investigated the possibility that the effect of aspirin is related to an antiplatelet mode of action. METHODS: In this study, we utilize the zebrafish-Mycobacterium marinum model to show mycobacteria drive host hemostasis through the formation of granulomas. Treatment of infected zebrafish with aspirin markedly reduced mycobacterial burden. This effect is reproduced by treatment with platelet-specific glycoprotein IIb/IIIa inhibitors demonstrating a detrimental role for infection-induced thrombocyte activation. RESULTS: We find that the reduction in mycobacterial burden is dependent on macrophages and granuloma formation, providing the first in vivo experimental evidence that infection-induced platelet activation compromises protective host immunity to mycobacterial infection. CONCLUSIONS: Our study illuminates platelet activation as an efficacious target of aspirin, a widely available and affordable host-directed therapy candidate for tuberculosis.

Modulation of Roquin Function in Myeloid Cells Reduces Mycobacterium tuberculosis-Induced Inflammation.

Damaging inflammation is a hallmark of Mycobacterium tuberculosis infection, and understanding how this is regulated is important for the development of new therapies to limit excessive inflammation. The E3 ubiquitin ligase, Roquin, is involved in immune regulation; however, its role in immunity to M. tuberculosis is unknown. To address this, we infected mice with a point mutation in Roquin1/Rc3h1 (sanroque). Aerosol-infected sanroque mice showed enhanced control of M. tuberculosis infection associated with delayed bacterial dissemination and upregulated TNF production in the lungs after 2 wk. However, this early control of infection was not maintained, and by 8 wk postinfection sanroque mice demonstrated an increased bacterial burden and dysregulated inflammation in the lungs. As the inflammation in the lungs of the sanroque mice could have been influenced by emerging autoimmune conditions that are characteristic of the mice aging, the function of Roquin was examined in immune cell subsets in the absence of autoimmune complications. M. bovis bacillus Calmette-Guérin-primed sanroque T cells transferred into Rag1-/- mice provided equivalent protection in the spleen and liver. Interestingly, the transfer of mycobacteria-specific (P25 CD4+ TCR transgenic) wild-type spleen cells into sanroqueRag1-/- mice actually led to enhanced protection with reduced bacterial load, decreased chemokine expression, and reduced inflammation in the lungs compared with transfers into Rag1-/- mice expressing intact Roquin. These studies suggest that modulation of Roquin in myeloid cells may reduce both inflammation and bacterial growth during the chronic phase of M. tuberculosis infection.