Blood RNA biomarkers for tuberculosis screening in people living with HIV before antiretroviral therapy initiation: a diagnostic accuracy study.

BACKGROUND: Undiagnosed tuberculosis remains a major threat for people living with HIV. Multiple blood transcriptomic biomarkers have shown promise for tuberculosis diagnosis. We sought to evaluate their diagnostic accuracy and clinical utility for systematic pre-antiretroviral therapy (ART) tuberculosis screening. METHODS: We enrolled consecutive adults (age ≥18 years) referred to start ART at a community health centre in Cape Town, South Africa, irrespective of symptoms. Sputa were obtained (using induction if required) for two liquid cultures. Whole-blood RNA samples underwent transcriptional profiling using a custom Nanostring gene panel. We measured the diagnostic accuracy of seven candidate RNA signatures (one single gene biomarker [BATF2] and six multigene biomarkers) for the reference standard of Mycobacterium tuberculosis culture status, using area under the receiver-operating characteristic curve (AUROC) analysis, and sensitivity and specificity at prespecified thresholds (two standard scores above the mean of healthy controls; Z2). Clinical utility was assessed by calculating net benefit in decision curve analysis. We compared performance with C-reactive protein (CRP; threshold ≥5 mg/L), WHO four-symptom screen (W4SS), and the WHO target product profile for tuberculosis triage tests. FINDINGS: A total of 707 people living with HIV (407 [58%] female and 300 [42%] male) were included, with median CD4 count 306 cells per mm3 (IQR 184-486). Of 676 participants with available sputum culture results, 89 (13%) had culture-confirmed tuberculosis. The seven RNA signatures were moderately to highly correlated (Spearman rank coefficients 0·42-0·93) and discriminated tuberculosis culture positivity with similar AUROCs (0·73-0·80), but none statistically better than CRP (AUROC 0·78, 95% CI 0·72-0·83). Diagnostic accuracy was similar across CD4 count strata, but lower among participants with negative W4SS (AUROCs 0·56-0·65) compared with positive (AUROCs 0·75-0·84). The RNA biomarker with the highest AUROC point estimate was a four-gene signature (Suliman4; AUROC 0·80, 95% CI 0·75-0·86), with sensitivity 83% (95% CI 74-90) and specificity 59% (55-63) at the Z2 threshold. In decision curve analysis, Suliman4 and CRP had similar clinical utility to guide confirmatory tuberculosis testing, but both had higher net benefit than W4SS. In exploratory analyses, an approach combining CRP (≥5 mg/L) and Suliman4 (≥Z2) had sensitivity of 80% (70-87), specificity of 70% (66-74), and higher net benefit than either biomarker alone. INTERPRETATION: RNA biomarkers showed better clinical utility to guide confirmatory tuberculosis testing for people living with HIV before ART initiation than symptom-based screening, but their performance did not exceed that of CRP and fell short of WHO recommended targets. Interferon-independent approaches might be required to improve accuracy of host-response biomarkers to support tuberculosis screening before ART initiation. FUNDING: South African Medical Research Council, European and Developing Countries Clinical Trials Partnership 2, National Institutes of Health National Institute of Allergy and Infectious Diseases, The Wellcome Trust, National Institute for Health and Care Research, Royal College of Physicians London.

Blood RNA biomarkers for tuberculosis screening in people living with HIV prior to anti-retroviral therapy initiation: A diagnostic accuracy study.

Background: Undiagnosed tuberculosis (TB) remains a major threat for people living with HIV (PLHIV). Multiple blood transcriptomic biomarkers have shown promise for TB diagnosis. We sought to evaluate their diagnostic accuracy and clinical utility for systematic pre-antiretroviral therapy (ART) TB screening. Methods: We enrolled consecutive adults referred to start ART at a community health centre in Cape Town, South Africa, irrespective of symptoms. Sputa were obtained (using induction if required) for two liquid cultures. Whole-blood RNA samples underwent transcriptional profiling using a custom Nanostring gene-panel. We measured the diagnostic accuracy of seven candidate RNA biomarkers for the reference standard of Mycobacterium tuberculosis culture status, using area under the receiver-operating characteristic curve (AUROC) analysis, and sensitivity/specificity at pre-specified thresholds (two standard scores above the mean of healthy controls; Z2). Clinical utility was assessed using decision curve analysis. We compared performance to CRP (threshold ≥5mg/L), World Health Organisation (WHO) four-symptom screen (W4SS) and the WHO target product profile for TB triage tests. Results: A total of 707 PLHIV were included, with median CD4 count 306 cells/mm3. Of 676 with available sputum culture results, 89 (13%) had culture-confirmed TB. The seven RNA biomarkers were moderately to highly correlated (Spearman rank coefficients 0.42-0.93) and discriminated TB culture-positivity with similar AUROCs (0.73-0.80), but none statistically better than CRP (AUROC 0.78; 95% CI 0.72-0.83). Diagnostic accuracy was similar across CD4 count strata, but lower among W4SS-negative (AUROCs 0.56-0.65) compared to W4SS-positive participants (AUROCs 0.75-0.84). The RNA biomarker with highest AUROC point estimate was a 4-gene signature (Suliman4; AUROC 0.80; 95% CI 0.75-0.86), with sensitivity 0.83 (0.74-0.90) and specificity 0.59 (0.55-0.63) at Z2 threshold. In decision curve analysis, Suliman4 and CRP had similar clinical utility to guide confirmatory TB testing, but both had higher net benefit than W4SS. In exploratory analyses, an approach combining CRP (≥5mg/L) and Suliman4 (≥Z2) had sensitivity of 0.80 (0.70-0.87), specificity of 0.70 (0.66-0.74) and higher net benefit than either biomarker alone. Interpretation: RNA biomarkers showed better clinical utility to guide confirmatory TB testing for PLHIV prior to ART initiation than symptom-based screening, but their performance did not exceed that of CRP, and fell short of WHO recommended targets. Interferon-independent approaches may be required to improve accuracy of host-response biomarkers to support TB screening pre-ART initiation. Funding: South African MRC, EDCTP2, NIH/NIAID, Wellcome Trust, NIHR, Royal College of Physicians London.

Large clones of pre-existing T cells drive early immunity against SARS-COV-2 and LCMV infection.

T cell responses precede antibody and may provide early control of infection. We analyzed the clonal basis of this rapid response following SARS-COV-2 infection. We applied T cell receptor (TCR) sequencing to define the trajectories of individual T cell clones immediately. In SARS-COV-2 PCR+ individuals, a wave of TCRs strongly but transiently expand, frequently peaking the same week as the first positive PCR test. These expanding TCR CDR3s were enriched for sequences functionally annotated as SARS-COV-2 specific. Epitopes recognized by the expanding TCRs were highly conserved between SARS-COV-2 strains but not with circulating human coronaviruses. Many expanding CDR3s were present at high frequency in pre-pandemic repertoires. Early response TCRs specific for lymphocytic choriomeningitis virus epitopes were also found at high frequency in the preinfection naive repertoire. High-frequency naive precursors may allow the T cell response to respond rapidly during the crucial early phases of acute viral infection.

Multi epitope vaccine candidate design against Streptococcus pneumonia.

Streptococcus pneumonia, the causative agent of sepsis, meningitis and pneumonia, is held responsible for causing invasive diseases predominantly in children along with adults from both developing and developed countries. The available vaccines coverage in the context of different serotypes is limited and emergence of non-vaccine serotypes could further emerge as a threat in future. Advanced immunoinformatics tools have been used for developing a multi epitope subunit vaccine. In the current study we have subjected these four surface antigenic proteins Ply, PsaA, PspA and PspK to construct vaccine designs. We have predicted different B-cell and T-cell epitopes by using NetCTL 1.2, IEDB (Immune Epitope Databases) and ABCpred. An adjuvant (griselimycin) has been added to the vaccine construct sequence in order to improve its immunogenicity. The vaccine construct has been evaluated for its antigenicity, allergenicity, toxicity and different physio-chemical properties. The bioinformatic tools have been used for prediction, refinement and validation of the 3 D structure. Further, the vaccine structure has been docked with a toll-like receptor (TLR-4) by ClusPro 2.0. In conclusion, the proposed multi-epitope vaccine designs could potentially activate both humoral and cellular immune responses and has a potential to be a vaccine candidate against S.pneumoniae, and requires experimental validation for ensuring immunogenicity and safety profile.Communicated by Ramaswamy H. Sarma.

Gefitinib Inhibits Rifampicin-Induced CYP3A4 Gene Expression in Human Hepatocytes.

During multidrug combination chemotherapy, activation of the nuclear receptor and the transcription factor human pregnane xenobiotic receptor (hPXR) has been shown to play a role in the development of chemoresistance. Mechanistically, this could occur due to the cancer drug activation of hPXR and the subsequent upregulation of hPXR target genes such as the drug metabolism enzyme, cytochrome P450 3A4 (CYP3A4). In the context of hPXR-mediated drug resistance, hPXR antagonists would be useful adjuncts to PXR-activating chemotherapy. However, there are currently no clinically approved hPXR antagonists in the market. Gefitinib (GEF), a tyrosine kinase inhibitor used for the treatment of advanced non-small-cell lung cancer and effectively used in combinational chemotherapy treatments, is a promising candidate owing to its hPXR ligand-like features. We, therefore, investigated whether GEF would act as an hPXR antagonist when combined with a known hPXR agonist, rifampicin (RIF). At therapeutically relevant concentrations, GEF successfully inhibited the RIF-induced upregulation of endogenous CYP3A4 gene expression in human primary hepatocytes and human hepatocells. Additionally, GEF inhibited the RIF induction of hPXR-mediated CYP3A4 promoter activity in HepG2 human liver carcinoma cells. The computational modeling of molecular docking predicted that GEF could bind to multiple sites on hPXR including the ligand-binding pocket, allowing for potential as a direct antagonist as well as an allosteric inhibitor. Indeed, GEF bound to the ligand-binding domain of the hPXR in cell-free assays, suggesting that GEF directly interacts with the hPXR. Taken together, our results suggest that GEF, at its clinically relevant therapeutic concentration, can antagonize the hPXR agonist-induced CYP3A4 gene expression in human hepatocytes. Thus, GEF could be a potential candidate for use in combinational chemotherapies to combat hPXR agonist-induced chemoresistance. Further studies are warranted to determine whether GEF has sufficient hPXR inhibitor abilities to overcome the hPXR agonist-induced chemoresistance.

Immune boosting by B.1.1.529 (Omicron) depends on previous SARS-CoV-2 exposure.

The Omicron, or Pango lineage B.1.1.529, variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) carries multiple spike mutations with high transmissibility and partial neutralizing antibody (nAb) escape. Vaccinated individuals show protection against severe disease, often attributed to primed cellular immunity. We investigated T and B cell immunity against B.1.1.529 in triple BioNTech BNT162b2 messenger RNA-vaccinated health care workers (HCWs) with different SARS-CoV-2 infection histories. B and T cell immunity against previous variants of concern was enhanced in triple-vaccinated individuals, but the magnitude of T and B cell responses against B.1.1.529 spike protein was reduced. Immune imprinting by infection with the earlier B.1.1.7 (Alpha) variant resulted in less durable binding antibody against B.1.1.529. Previously infection-naïve HCWs who became infected during the B.1.1.529 wave showed enhanced immunity against earlier variants but reduced nAb potency and T cell responses against B.1.1.529 itself. Previous Wuhan Hu-1 infection abrogated T cell recognition and any enhanced cross-reactive neutralizing immunity on infection with B.1.1.529.

Structure-Based Discovery of Lipoteichoic Acid Synthase Inhibitors.

Lipoteichoic acid synthase (LtaS) is a key enzyme for the cell wall biosynthesis of Gram-positive bacteria. Gram-positive bacteria that lack lipoteichoic acid (LTA) exhibit impaired cell division and growth defects. Thus, LtaS appears to be an attractive antimicrobial target. The pharmacology around LtaS remains largely unexplored with only two small-molecule LtaS inhibitors reported, namely "compound 1771" and the Congo red dye. Structure-based drug discovery efforts against LtaS remain unattempted due to the lack of an inhibitor-bound structure of LtaS. To address this, we combined the use of a molecular docking technique with molecular dynamics (MD) simulations to model a plausible binding mode of compound 1771 to the extracellular catalytic domain of LtaS (eLtaS). The model was validated using alanine mutagenesis studies combined with isothermal titration calorimetry. Additionally, lead optimization driven by our computational model resulted in an improved version of compound 1771, namely, compound 4 which showed greater affinity for binding to eLtaS than compound 1771 in biophysical assays. Compound 4 reduced LTA production in S. aureus dose-dependently, induced aberrant morphology as seen for LTA-deficient bacteria, and significantly reduced bacteria titers in the lung of mice infected with S. aureus. Analysis of our MD simulation trajectories revealed the possible formation of a transient cryptic pocket in eLtaS. Virtual screening (VS) against the cryptic pocket led to the identification of a new class of inhibitors that could potentiate ß-lactams against methicillin-resistant S. aureus. Our overall workflow and data should encourage further drug design campaign against LtaS. Finally, our work reinforces the importance of considering protein conformational flexibility to a successful VS endeavor.

Rapid synchronous type 1 IFN and virus-specific T cell responses characterize first wave non-severe SARS-CoV-2 infections.

Effective control of SARS-CoV-2 infection on primary exposure may reveal correlates of protective immunity to future variants, but we lack insights into immune responses before or at the time virus is first detected. We use blood transcriptomics, multiparameter flow cytometry, and T cell receptor (TCR) sequencing spanning the time of incident non-severe infection in unvaccinated virus-naive individuals to identify rapid type 1 interferon (IFN) responses common to other acute respiratory viruses and cell proliferation responses that discriminate SARS-CoV-2 from other viruses. These peak by the time the virus is first detected and sometimes precede virus detection. Cell proliferation is most evident in CD8 T cells and associated with specific expansion of SARS-CoV-2-reactive TCRs, in contrast to virus-specific antibodies, which lag by 1-2 weeks. Our data support a protective role for early type 1 IFN and CD8 T cell responses, with implications for development of universal T cell vaccines.

Pre-existing polymerase-specific T cells expand in abortive seronegative SARS-CoV-2.

Individuals with potential exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) do not necessarily develop PCR or antibody positivity, suggesting that some individuals may clear subclinical infection before seroconversion. T cells can contribute to the rapid clearance of SARS-CoV-2 and other coronavirus infections1-3. Here we hypothesize that pre-existing memory T cell responses, with cross-protective potential against SARS-CoV-2 (refs. 4-11), would expand in vivo to support rapid viral control, aborting infection. We measured SARS-CoV-2-reactive T cells, including those against the early transcribed replication-transcription complex (RTC)12,13, in intensively monitored healthcare workers (HCWs) who tested repeatedly negative according to PCR, antibody binding and neutralization assays (seronegative HCWs (SN-HCWs)). SN-HCWs had stronger, more multispecific memory T cells compared with a cohort of unexposed individuals from before the pandemic (prepandemic cohort), and these cells were more frequently directed against the RTC than the structural-protein-dominated responses observed after detectable infection (matched concurrent cohort). SN-HCWs with the strongest RTC-specific T cells had an increase in IFI27, a robust early innate signature of SARS-CoV-2 (ref. 14), suggesting abortive infection. RNA polymerase within RTC was the largest region of high sequence conservation across human seasonal coronaviruses (HCoV) and SARS-CoV-2 clades. RNA polymerase was preferentially targeted (among the regions tested) by T cells from prepandemic cohorts and SN-HCWs. RTC-epitope-specific T cells that cross-recognized HCoV variants were identified in SN-HCWs. Enriched pre-existing RNA-polymerase-specific T cells expanded in vivo to preferentially accumulate in the memory response after putative abortive compared to overt SARS-CoV-2 infection. Our data highlight RTC-specific T cells as targets for vaccines against endemic and emerging Coronaviridae.

Blood transcriptional biomarkers of acute viral infection for detection of pre-symptomatic SARS-CoV-2 infection: a nested, case-control diagnostic accuracy study.

BACKGROUND: We hypothesised that host-response biomarkers of viral infections might contribute to early identification of individuals infected with SARS-CoV-2, which is critical to breaking the chains of transmission. We aimed to evaluate the diagnostic accuracy of existing candidate whole-blood transcriptomic signatures for viral infection to predict positivity of nasopharyngeal SARS-CoV-2 PCR testing. METHODS: We did a nested case-control diagnostic accuracy study among a prospective cohort of health-care workers (aged ≥18 years) at St Bartholomew's Hospital (London, UK) undergoing weekly blood and nasopharyngeal swab sampling for whole-blood RNA sequencing and SARS-CoV-2 PCR testing, when fit to attend work. We identified candidate blood transcriptomic signatures for viral infection through a systematic literature search. We searched MEDLINE for articles published between database inception and Oct 12, 2020, using comprehensive MeSH and keyword terms for "viral infection", "transcriptome", "biomarker", and "blood". We reconstructed signature scores in blood RNA sequencing data and evaluated their diagnostic accuracy for contemporaneous SARS-CoV-2 infection, compared with the gold standard of SARS-CoV-2 PCR testing, by quantifying the area under the receiver operating characteristic curve (AUROC), sensitivities, and specificities at a standardised Z score of at least 2 based on the distribution of signature scores in test-negative controls. We used pairwise DeLong tests compared with the most discriminating signature to identify the subset of best performing biomarkers. We evaluated associations between signature expression, viral load (using PCR cycle thresholds), and symptom status visually and using Spearman rank correlation. The primary outcome was the AUROC for discriminating between samples from participants who tested negative throughout the study (test-negative controls) and samples from participants with PCR-confirmed SARS-CoV-2 infection (test-positive participants) during their first week of PCR positivity. FINDINGS: We identified 20 candidate blood transcriptomic signatures of viral infection from 18 studies and evaluated their accuracy among 169 blood RNA samples from 96 participants over 24 weeks. Participants were recruited between March 23 and March 31, 2020. 114 samples were from 41 participants with SARS-CoV-2 infection, and 55 samples were from 55 test-negative controls. The median age of participants was 36 years (IQR 27-47) and 69 (72%) of 96 were women. Signatures had little overlap of component genes, but were mostly correlated as components of type I interferon responses. A single blood transcript for IFI27 provided the highest accuracy for discriminating between test-negative controls and test-positive individuals at the time of their first positive SARS-CoV-2 PCR result, with AUROC of 0·95 (95% CI 0·91-0·99), sensitivity 0·84 (0·70-0·93), and specificity 0·95 (0·85-0·98) at a predefined threshold (Z score >2). The transcript performed equally well in individuals with and without symptoms. Three other candidate signatures (including two to 48 transcripts) had statistically equivalent discrimination to IFI27 (AUROCs 0·91-0·95). INTERPRETATION: Our findings support further urgent evaluation and development of blood IFI27 transcripts as a biomarker for early phase SARS-CoV-2 infection for screening individuals at high risk of infection, such as contacts of index cases, to facilitate early case isolation and early use of antiviral treatments as they emerge. FUNDING: Barts Charity, Wellcome Trust, and National Institute of Health Research.

Prognostic implications of troponin T variations in inherited cardiomyopathies using systems biology.

The cardiac troponin T variations have often been used as an example of the application of clinical genotyping for prognostication and risk stratification measures for the management of patients with a family history of sudden cardiac death or familial cardiomyopathy. Given the disparity in patient outcomes and therapy options, we investigated the impact of variations on the intermolecular interactions across the thin filament complex as an example of an unbiased systems biology method to better define clinical prognosis to aid future management options. We present a novel unbiased dynamic model to define and analyse the functional, structural and physico-chemical consequences of genetic variations among the troponins. This was subsequently integrated with clinical data from accessible global multi-centre systematic reviews of familial cardiomyopathy cases from 106 articles of the literature: 136 disease-causing variations pertaining to 981 global clinical cases. Troponin T variations showed distinct pathogenic hotspots for dilated and hypertrophic cardiomyopathies; considering the causes of cardiovascular death separately, there was a worse survival in terms of sudden cardiac death for patients with a variation at regions 90-129 and 130-179 when compared to amino acids 1-89 and 200-288. Our data support variations among 90-130 as being a hotspot for sudden cardiac death and the region 131-179 for heart failure death/transplantation outcomes wherein the most common phenotype was dilated cardiomyopathy. Survival analysis into regions of high risk (regions 90-129 and 130-180) and low risk (regions 1-89 and 200-288) was significant for sudden cardiac death (p = 0.011) and for heart failure death/transplant (p = 0.028). Our integrative genomic, structural, model from genotype to clinical data integration has implications for enhancing clinical genomics methodologies to improve risk stratification.

Detection of Mycobacterium tuberculosis complex DNA in CD34-positive peripheral blood mononuclear cells of asymptomatic tuberculosis contacts: an observational study.

BACKGROUND: Haematopoietic stem cells expressing the CD34 surface marker have been posited as a niche for Mycobacterium tuberculosis complex bacilli during latent tuberculosis infection. Our aim was to determine whether M tuberculosis complex DNA is detectable in CD34-positive peripheral blood mononuclear cells (PBMCs) isolated from asymptomatic adults living in a setting with a high tuberculosis burden. METHODS: We did a cross-sectional study in Ethiopia between Nov 22, 2017, and Jan 10, 2019. Digital PCR (dPCR) was used to determine whether M tuberculosis complex DNA was detectable in PBMCs isolated from 100 mL blood taken from asymptomatic adults with HIV infection or a history of recent household or occupational exposure to an index case of human or bovine tuberculosis. Participants were recruited from HIV clinics, tuberculosis clinics, and cattle farms in and around Addis Ababa. A nested prospective study was done in a subset of HIV-infected individuals to evaluate whether administration of isoniazid preventive therapy was effective in clearing M tuberculosis complex DNA from PBMCs. Follow-up was done between July 20, 2018, and Feb 13, 2019. QuantiFERON-TB Gold assays were also done on all baseline and follow-up samples. FINDINGS: Valid dPCR data (ie, droplet counts >10 000 per well) were available for paired CD34-positive and CD34-negative PBMC fractions from 197 (70%) of 284 participants who contributed data to cross-sectional analyses. M tuberculosis complex DNA was detected in PBMCs of 156 of 197 participants with valid dPCR data (79%, 95% CI 74-85). It was more commonly present in CD34-positive than in CD34-negative fractions (154 [73%] of 197 vs 46 [23%] of 197; p<0·0001). Prevalence of dPCR-detected M tuberculosis complex DNA did not differ between QuantiFERON-negative and QuantiFERON-positive participants (77 [78%] of 99 vs 79 [81%] of 98; p=0·73), but it was higher in HIV-infected than in HIV-uninfected participants (67 [89%] of 75 vs 89 [73%] of 122, p=0·0065). By contrast, the proportion of QuantiFERON-positive participants was lower in HIV-infected than in HIV-uninfected participants (25 [33%] of 75 vs 73 [60%] of 122; p<0·0001). Administration of isoniazid preventive therapy reduced the prevalence of dPCR-detected M tuberculosis complex DNA from 41 (95%) of 43 HIV-infected individuals at baseline to 23 (53%) of 43 after treatment (p<0·0001), but it did not affect the prevalence of QuantiFERON positivity (17 [40%] of 43 at baseline vs 13 [30%] of 43 after treatment; p=0·13). INTERPRETATION: We report a novel molecular microbiological biomarker of latent tuberculosis infection with properties that are distinct from those of a commercial interferon-γ release assay. Our findings implicate the bone marrow as a niche for M tuberculosis in latently infected individuals. Detection of M tuberculosis complex DNA in PBMCs has potential applications in the diagnosis of latent tuberculosis infection, in monitoring response to preventive therapy, and as an outcome measure in clinical trials of interventions to prevent or treat latent tuberculosis infection. FUNDING: UK Medical Research Council.

Exaggerated IL-17A activity in human in vivo recall responses discriminates active tuberculosis from latent infection and cured disease.

Host immune responses at the site of Mycobacterium tuberculosis infection can mediate pathogenesis of tuberculosis (TB) and onward transmission of infection. We hypothesized that pathological immune responses would be enriched at the site of host-pathogen interactions modeled by a standardized tuberculin skin test (TST) challenge in patients with active TB compared to those without disease, and interrogated immune responses by genome-wide transcriptional profiling. We show exaggerated interleukin-17A (IL-17A) and T helper 17 (TH17) responses among 48 individuals with active TB compared to 191 with latent TB infection, associated with increased neutrophil recruitment and matrix metalloproteinase-1 expression, both involved in TB pathogenesis. Curative antimicrobial treatment reversed these observed changes. Increased IL-1ß and IL-6 responses to mycobacterial stimulation were evident both in circulating monocytes and in molecular changes at the site of TST in individuals with active TB, supporting a model in which monocyte-derived IL-1ß and IL-6 promote TH17 differentiation within tissues. Modulation of these cytokine pathways may provide a rational strategy for host-directed therapy in active TB.

Discordant neutralizing antibody and T cell responses in asymptomatic and mild SARS-CoV-2 infection.

Understanding the nature of immunity following mild/asymptomatic infection with SARS-CoV-2 is crucial to controlling the pandemic. We analyzed T cell and neutralizing antibody responses in 136 healthcare workers (HCW) 16-18 weeks after United Kingdom lockdown, 76 of whom had mild/asymptomatic SARS-CoV-2 infection captured by serial sampling. Neutralizing antibodies (nAb) were present in 89% of previously infected HCW. T cell responses tended to be lower following asymptomatic infection than in those reporting case-definition symptoms of COVID-19, while nAb titers were maintained irrespective of symptoms. T cell and antibody responses were sometimes discordant. Eleven percent lacked nAb and had undetectable T cell responses to spike protein but had T cells reactive with other SARS-CoV-2 antigens. Our findings suggest that the majority of individuals with mild or asymptomatic SARS-CoV-2 infection carry nAb complemented by multispecific T cell responses at 16-18 weeks after mild or asymptomatic SARS-CoV-2 infection.

Author Correction: Development of a diagnostic compatible BCG vaccine against Bovine tuberculosis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Proposed model of the Dictyostelium cAMP receptors bound to cAMP.

3',5'-cyclic adenosine monophosphate (cAMP) is well known as a ubiquitous intracellular messenger regulating a diverse array of cellular processes. However, for a group of social amoebae or Dictyostelia undergoing starvation, intracellular cAMP is secreted in a pulsatile manner to their exterior. This then uniquely acts as a first messenger, triggering aggregation of the starving amoebae followed by their developmental progression towards multicellular fruiting bodies formation. Such developmental signalling for extracellularly-acting cAMP is well studied in the popular dictyostelid, Dictyostelium discoideum, and is mediated by a distinct family ('class E') of G protein-coupled receptors (GPCRs) collectively designated as the cAMP receptors (cARs). Whilst the biochemical aspects of these receptors are well characterised, little is known about their overall 3D architecture and structural basis for cAMP recognition and subtype-dependent changes in binding affinity. Using a ligand docking-guided homology modelling approach, we hereby present for the first time, plausible models of active forms of the cARs from D. discoideum. Our models highlight some structural features that may underlie the differential affinities of cAR isoforms for cAMP binding and also suggest few residues that may play important roles for the activation mechanism of this GPCR family.

Antimigraine Drug Avitriptan Is a Ligand and Agonist of Human Aryl Hydrocarbon Receptor That Induces CYP1A1 in Hepatic and Intestinal Cells.

The efforts for therapeutic targeting of the aryl hydrocarbon receptor (AhR) have emerged in recent years. We investigated the effects of available antimigraine triptan drugs, having an indole core in their structure, on AhR signaling in human hepatic and intestinal cells. Activation of AhR in reporter gene assays was observed for Avitriptan and to a lesser extent for Donitriptan, while other triptans were very weak or no activators of AhR. Using competitive binding assay and by homology docking, we identified Avitriptan as a low-affinity ligand of AhR. Avitriptan triggered nuclear translocation of AhR and increased binding of AhR in CYP1A1 promotor DNA, as revealed by immune-fluorescence microscopy and chromatin immune-precipitation assay, respectively. Strong induction of CYP1A1 mRNA was achieved by Avitriptan in wild type but not in AhR-knockout, immortalized human hepatocytes, implying that induction of CYP1A1 is AhR-dependent. Increased levels of CYP1A1 mRNA by Avitriptan were observed in human colon carcinoma cells LS180 but not in primary cultures of human hepatocytes. Collectively, we show that Avitriptan is a weak ligand and activator of human AhR, which induces the expression of CYP1A1 in a cell-type specific manner. Our data warrant the potential off-label therapeutic application of Avitriptan as an AhR-agonist drug.

Targeting the pregnane X receptor using microbial metabolite mimicry.

The human PXR (pregnane X receptor), a master regulator of drug metabolism, has essential roles in intestinal homeostasis and abrogating inflammation. Existing PXR ligands have substantial off-target toxicity. Based on prior work that established microbial (indole) metabolites as PXR ligands, we proposed microbial metabolite mimicry as a novel strategy for drug discovery that allows exploiting previously unexplored parts of chemical space. Here, we report functionalized indole derivatives as first-in-class non-cytotoxic PXR agonists as a proof of concept for microbial metabolite mimicry. The lead compound, FKK6 (Felix Kopp Kortagere 6), binds directly to PXR protein in solution, induces PXR-specific target gene expression in cells, human organoids, and mice. FKK6 significantly represses pro-inflammatory cytokine production cells and abrogates inflammation in mice expressing the human PXR gene. The development of FKK6 demonstrates for the first time that microbial metabolite mimicry is a viable strategy for drug discovery and opens the door to underexploited regions of chemical space.

Mycobacterium bovis uses the ESX-1 Type VII secretion system to escape predation by the soil-dwelling amoeba Dictyostelium discoideum.

Mycobacterium bovis is the causative agent of bovine tuberculosis and the predominant cause of zoonotic tuberculosis in people. Bovine tuberculosis occurs in farmed cattle but also in a variety of wild animals, which form a reservoir of infection. Although direct transmission of tuberculosis occurs between mammals, the low frequency of contact between different host species and abundant shedding of bacilli by infected animals suggests an infectious route via environmental contamination. Other intracellular pathogens that transmit via the environment deploy strategies to survive or exploit predation by environmental amoebae. To explore if M. bovis has this capability, we investigated its interactions with the soil and dung-dwelling amoeba, Dictyostelium discoideum. We demonstrated that M. bovis evades phagocytosis and destruction by D. discoideum and actively transits through the amoeba using the ESX-1 Type VII Secretion System as part of a programme of mechanisms, many of which have been co-opted as virulence factors in the mammalian host. This capacity of M. bovis to utilise an environmental stage between mammalian hosts may enhance its transmissibility. In addition, our data provide molecular evidence to support an evolutionary role for amoebae as training grounds for the pathogenic M. tuberculosis complex.

Development of a diagnostic compatible BCG vaccine against Bovine tuberculosis.

Bovine tuberculosis (BTB) caused by Mycobacterium bovis remains a major problem in both the developed and developing countries. Control of BTB in the UK is carried out by test and slaughter of infected animals, based primarily on the tuberculin skin test (PPD). Vaccination with the attenuated strain of the M. bovis pathogen, BCG, is not used to control bovine tuberculosis in cattle at present, due to its variable efficacy and because it interferes with the PPD test. Diagnostic tests capable of Differentiating Infected from Vaccinated Animals (DIVA) have been developed that detect immune responses to M. bovis antigens absent in BCG; but these are too expensive and insufficiently sensitive to be used for BTB control worldwide. To address these problems we aimed to generate a synergistic vaccine and diagnostic approach that would permit the vaccination of cattle without interfering with the conventional PPD-based surveillance. The approach was to widen the pool of M. bovis antigens that could be used as DIVA targets, by identifying antigenic proteins that could be deleted from BCG without affecting the persistence and protective efficacy of the vaccine in cattle. Using transposon mutagenesis we identified genes that were essential and those that were non-essential for persistence in bovine lymph nodes. We then inactivated selected immunogenic, but non-essential genes in BCG Danish to create a diagnostic-compatible triple knock-out ΔBCG TK strain. The protective efficacy of the ΔBCG TK was tested in guinea pigs experimentally infected with M. bovis by aerosol and found to be equivalent to wild-type BCG. A complementary diagnostic skin test was developed with the antigenic proteins encoded by the deleted genes which did not cross-react in vaccinated or in uninfected guinea pigs. This study demonstrates the functionality of a new and improved BCG strain which retains its protective efficacy but is diagnostically compatible with a novel DIVA skin test that could be implemented in control programmes.