An immunostimulatory glycolipid that blocks SARS-CoV-2, RSV, and influenza infections in vivo.

Prophylactic vaccines for SARS-CoV-2 have lowered the incidence of severe COVID-19, but emergence of viral variants that are antigenically distinct from the vaccine strains are of concern and additional, broadly acting preventive approaches are desirable. Here, we report on a glycolipid termed 7DW8-5 that exploits the host innate immune system to enable rapid control of viral infections in vivo. This glycolipid binds to CD1d on antigen-presenting cells and thereby stimulates NKT cells to release a cascade of cytokines and chemokines. The intranasal administration of 7DW8-5 prior to virus exposure significantly blocked infection by three different authentic variants of SARS-CoV-2, as well as by respiratory syncytial virus and influenza virus, in mice or hamsters. We also found that this protective antiviral effect is both host-directed and mechanism-specific, requiring both the CD1d molecule and interferon-[Formula: see text]. A chemical compound like 7DW8-5 that is easy to administer and cheap to manufacture may be useful not only in slowing the spread of COVID-19 but also in responding to future pandemics long before vaccines or drugs are developed.

New, fast, and precise method of COVID-19 detection in nasopharyngeal and tracheal aspirate samples combining optical spectroscopy and machine learning.

Fast, precise, and low-cost diagnostic testing to identify persons infected with SARS-CoV-2 virus is pivotal to control the global pandemic of COVID-19 that began in late 2019. The gold standard method of diagnostic recommended is the RT-qPCR test. However, this method is not universally available, and is time-consuming and requires specialized personnel, as well as sophisticated laboratories. Currently, machine learning is a useful predictive tool for biomedical applications, being able to classify data from diverse nature. Relying on the artificial intelligence learning process, spectroscopic data from nasopharyngeal swab and tracheal aspirate samples can be used to leverage characteristic patterns and nuances in healthy and infected body fluids, which allows to identify infection regardless of symptoms or any other clinical or laboratorial tests. Hence, when new measurements are performed on samples of unknown status and the corresponding data is submitted to such an algorithm, it will be possible to predict whether the source individual is infected or not. This work presents a new methodology for rapid and precise label-free diagnosing of SARS-CoV-2 infection in clinical samples, which combines spectroscopic data acquisition and analysis via artificial intelligence algorithms. Our results show an accuracy of 85% for detection of SARS-CoV-2 in nasopharyngeal swab samples collected from asymptomatic patients or with mild symptoms, as well as an accuracy of 97% in tracheal aspirate samples collected from critically ill COVID-19 patients under mechanical ventilation. Moreover, the acquisition and processing of the information is fast, simple, and cheaper than traditional approaches, suggesting this methodology as a promising tool for biomedical diagnosis vis-à-vis the emerging and re-emerging viral SARS-CoV-2 variant threats in the future.

Taming the SARS-CoV-2-mediated proinflammatory response with BromAc®.

Introduction: In the present study, the impact of BromAc®, a specific combination of bromelain and acetylcysteine, on the SARS-CoV-2-specific inflammatory response was evaluated. Methods: An in vitro stimulation system was standardized using blood samples from 9 healthy donors, luminex assays and flow cytometry were performed. Results and discussion: BromAc® demonstrated robust anti-inflammatory activity in human peripheral blood cells upon SARS-CoV-2 viral stimuli, reducing the cytokine storm, composed of chemokines, growth factors, and proinflammatory and regulatory cytokines produced after short-term in vitro culture with the inactivated virus (iSARS-CoV-2). A combined reduction in vascular endothelial growth factor (VEGF) induced by SARS-CoV-2, in addition to steady-state levels of platelet recruitment-associated growth factor-PDGFbb, was observed, indicating that BromAc® may be important to reduce thromboembolism in COVID-19. The immunophenotypic analysis of the impact of BromAc® on leukocytes upon viral stimuli showed that BromAc® was able to downmodulate the populations of CD16+ neutrophils and CD14+ monocytes observed after stimulation with iSARS-CoV-2. Conversely, BromAc® treatment increased steady-state HLA-DR expression in CD14+ monocytes and preserved this activation marker in this subset upon iSARS-CoV-2 stimuli, indicating improved monocyte activation upon BromAc® treatment. Additionally, BromAc® downmodulated the iSARS-CoV-2-induced production of TNF-a by the CD19+ B-cells. System biology approaches, utilizing comprehensive correlation matrices and networks, showed distinct patterns of connectivity in groups treated with BromAc®, suggesting loss of connections promoted by the compound and by iSARS-CoV-2 stimuli. Negative correlations amongst proinflammatory axis and other soluble and cellular factors were observed in the iSARS-CoV-2 group treated with BromAc® as compared to the untreated group, demonstrating that BromAc® disengages proinflammatory responses and their interactions with other soluble factors and the axis orchestrated by SARS-CoV-2. Conclusion: These results give new insights into the mechanisms for the robust anti-inflammatory effect of BromAc® in the steady state and SARS-CoV-2-specific immune leukocyte responses, indicating its potential as a therapeutic strategy for COVID-19.

The role of mucosal-associated invariant T cells in visceral leishmaniasis.

Mucosal-associated invariant T (MAIT) cells are restricted by MR1 and are known to protect against bacterial and viral infections. Our understanding of the role of MAIT cells in parasitic infections, such as visceral leishmaniasis (VL) caused by protozoan parasites of Leishmania donovani, is limited. This study showed that in response to L. infantum, human peripheral blood MAIT cells from children with leishmaniasis produced TNF and IFN-γ in an MR1-dependent manner. The overall frequency of MAIT cells was inversely correlated with alanine aminotransferase levels, a specific marker of liver damage strongly associated with severe hepatic involvement in VL. In addition, there was a positive correlation between total protein levels and the frequency of IL-17A+ CD8+ MAIT cells, whereby reduced total protein levels are a marker of liver and kidney damage. Furthermore, the frequencies of IFN-γ+ and IL-10+ MAIT cells were inversely correlated with hemoglobin levels, a marker of severe anemia. In asymptomatic individuals and VL patients after treatment, MAIT cells also produced IL-17A, a cytokine signature associated with resistance to visceral leishmaniasis, suggesting that MAIT cells play important role in protecting against VL. In summary, these results broaden our understanding of MAIT-cell immunity to include protection against parasitic infections, with implications for MAIT-cell-based therapeutics and vaccines. At last, this study paves the way for the investigation of putative MAIT cell antigens that could exist in the context of Leishmania infection.

Timeline Kinetics of Systemic and Airway Immune Mediator Storm for Comprehensive Analysis of Disease Outcome in Critically Ill COVID-19 Patients.

In the present study, the levels of serum and airway soluble chemokines, pro-inflammatory/regulatory cytokines, and growth factors were quantified in critically ill COVID-19 patients (total n=286) at distinct time points (D0, D2-6, D7, D8-13 and D>14-36) upon Intensive Care Unit (ICU) admission. Augmented levels of soluble mediators were observed in serum from COVID-19 patients who progress to death. An opposite profile was observed in tracheal aspirate samples, indicating that systemic and airway microenvironment diverge in their inflammatory milieu. While a bimodal distribution was observed in the serum samples, a unimodal peak around D7 was found for most soluble mediators in tracheal aspirate samples. Systems biology tools further demonstrated that COVID-19 display distinct eccentric soluble mediator networks as compared to controls, with opposite profiles in serum and tracheal aspirates. Regardless the systemic-compartmentalized microenvironment, networks from patients progressing to death were linked to a pro-inflammatory/growth factor-rich, highly integrated center. Conversely, patients evolving to discharge exhibited networks of weak central architecture, with lower number of neighborhood connections and clusters of pro-inflammatory and regulatory cytokines. All in all, this investigation with robust sample size landed a comprehensive snapshot of the systemic and local divergencies composed of distinct immune responses driven by SARS-CoV-2 early on severe COVID-19.

Ex-vivo mucolytic and anti-inflammatory activity of BromAc in tracheal aspirates from COVID-19.

COVID-19 is a lethal disease caused by the pandemic SARS-CoV-2, which continues to be a public health threat. COVID-19 is principally a respiratory disease and is often associated with sputum retention and cytokine storm, for which there are limited therapeutic options. In this regard, we evaluated the use of BromAc®, a combination of Bromelain and Acetylcysteine (NAC). Both drugs present mucolytic effect and have been studied to treat COVID-19. Therefore, we sought to examine the mucolytic and anti-inflammatory effect of BromAc® in tracheal aspirate samples from critically ill COVID-19 patients requiring mechanical ventilation. METHOD: Tracheal aspirate samples from COVID-19 patients were collected following next of kin consent and mucolysis, rheometry and cytokine analysis using Luminex kit was performed. RESULTS: BromAc® displayed a robust mucolytic effect in a dose dependent manner on COVID-19 sputum ex vivo. BromAc® showed anti-inflammatory activity, reducing the action of cytokine storm, chemokines including MIP-1alpha, CXCL8, MIP-1b, MCP-1 and IP-10, and regulatory cytokines IL-5, IL-10, IL-13 IL-1Ra and total reduction for IL-9 compared to NAC alone and control. BromAc® acted on IL-6, demonstrating a reduction in G-CSF and VEGF-D at concentrations of 125 and 250 µg. CONCLUSION: These results indicate robust mucolytic and anti-inflammatory effect of BromAc® ex vivo in tracheal aspirates from critically ill COVID-19 patients, indicating its potential to be further assessed as pharmacological treatment for COVID-19.

A chimeric HLA-A2:ß2M:Ig fusion protein for the study of virus-specific CD8+ T-cells.

INTRODUCTION: The response mediated by CD8+ T-cells in the context of infection and vaccination has been thoroughly investigated and represents one of the most important branches that allow for the development of immunity against intracellular pathogens and, thus, the establishment of robust antiviral responses. However, there is a lack of methods to assess antigen-specific CD8+ T-cells. OBJECTIVE: Search for the ideal assays to assess the function of antigen-specific CD8+ T-cells. METHODS: In the present study a chimeric HLA-A2:ß2M:Ig fusion protein was produced, purified, and evaluated in functional CD8+ T-cell response studies using samples from Influenza A patients and humanized mice upon adenoviral vaccination. RESULTS: The HLA-A2:ß2M:Ig molecule, bound to immunodominant viral peptides by passive transfer, was able to induce robust antiviral CD8+ T-cell responses mediated by IFN-γ. The in vitro IFN-γ release assay using the chimeric HLA-A2:ß2M:Ig fusion protein detected bona fide human CD8+ T-cells, demonstrating superior production of IFN-γ by human CD8+ T-cells induced by Influenza A immunodominant GILGFVFTL peptide. Removal of antigen-presenting cells and CD8+ T-cell enrichment improved significantly the IFN-γ production. The chimeric HLA-A2:ß2M:Ig fusion protein also triggered HLA-A2-restricted CD8+ T-cell response in a humanized mouse model upon vaccination with adenovirus encoding HLA-A2-restricted HIV p24 antigen. The results strongly suggest the use of tailor-made assays for detecting HLA-A2-restricted CD8+ T-cell Responses in the Humanized Mouse Model. CONCLUSION: The chimeric HLA-A2:ß2M:Ig fusion protein-based assays provided a sensitive tool that may be paramount to measure virus-specific CD8+ T-cell response in a range of viral infections of clinical relevance.

Early serum biomarker networks in infants with distinct retinochoroidal lesion status of congenital toxoplasmosis.

The present study characterized the early changes in the serum chemokines/cytokine signatures and networks in infants with congenital-toxoplasmosis/(TOXO) as compared to non-infected-controls/(NI). TOXO were subgrouped according to the retinochoroidal lesion status as no-lesion/(NL), active-lesion/(ARL), active/cicatricial-lesion/(ACRL) and cicatricial-lesion/(CRL). The results showed that TOXO display prominent chemokine production mediated by IL-8/CXCL8, MIG/CXCL9, IP-10/CXCL10 and RANTES/CCL5. Additionally, TOXO is accompanied by mixed proinflammatory/regulatory cytokine pattern mediated by IL-6, IFN-γ, IL-4, IL-5 and IL-10. While TNF appears as a putative biomarker for NL and IFN-γ/IL-5 as immunological features for ARL, IL-10 emerges as a relevant mediator in ACRL/CRL. IL-8/CXCL8 and IP-10/CXCL10 are broad-spectrum indicators of ocular disease, whereas TNF is a NL biomarker, IFN-γ and MIG/CXCL9 point out to ARL; and IL-10 is highlighted as a genuine serum biomarker of ACRL/CRL. The network analysis demonstrated a broad chemokine/cytokine crosstalk with divergences in the molecular signatures in patients with different ocular lesions during congenital toxoplasmosis.

Flow cytometric-based protocols for assessing anti-MT-2 IgG1 reactivity: High-dimensional data handling to define predictors for clinical follow-up of Human T-cell Leukemia virus type-1 infection.

The present work provides an innovative methodological approach to assess the anti-HTLV-1 IgG1 reactivity with practical application in clinical laboratory. Serum from non-infected healthy controls (NI) and HTLV-1-infected patients, categorized as asymptomatic (AS), putatively progressing to HTLV-1 associated myelopathy/tropical spastic paraparesis - HAM/TSP (pHAM) or with clinical diagnosis of HAM/TSP (HT) were assayed in two-parallel flow cytometry platforms, referred as: Fix and Fix&Perm protocols. Operating-characteristics analysis indicated that a single pair of attributes ("serum dilution/cut-off") for Fix and Fix&Perm protocols presented excellent performance for the diagnosis of HTLV-1 infection. Conversely, Fix and Fix&Perm protocols displayed weak/moderate overall performances when applied with prognosis purposes of HTLV-1 infection. A panoramic snapshot provided by the reactivity boards revealed clearly the higher sensitivity of Fix&Perm protocol for detecting seropositivity for HT, suggesting that stepwise combinatory criteria would improve the global performance of using a single pair of attributes. Three data mining strategies were tested, including endpoint titer analysis, heatmap assemblage and decision tree analysis. Bi-dimensional heatmap analysis demonstrated that, while the clustering profile of NI vs HTLV-1+ revealed segregation in opposite poles, AS vs HT presented discrete segregation but still displaying an intertwined distribution pattern. The combination of methods for segregating AS from HT displayed a moderate but superior global accuracy (85.7%; LOOCV=71.4%). The comprehensive data analysis support that the combination of methods have improved the performance to the differential diagnosis of AS and HT, with direct association with laboratorial records, including serum cytokine levels and proviral load.