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Background/Objectives: The broad spectrum of clinical manifestations from SARS-COV-2 infection and observed risk factors for severe disease highlight the importance of understanding molecular mechanisms underlying SARS-CoV-2 associated disease pathogenesis. Research studies have identified a large number of host proteins that play roles in viral entry, innate immune response, or immune signalling during infection. The ability to interrogate subsets of these genes simultaneously within SARSCOV-2 infected samples is critical to understanding how their expression contribute to phenotypic variability of the disease caused by the pathogen. Method(s): 30 Nasopharyngeal swab were obtained and included SARS-CoV-2 infected and control samples. RNA was extracted, reverse transcribed and loaded onto flexible TaqMan array panels designed specifically for targeting the most cited genes related to SARS-COV-2 entry and restriction factors as well as cytokines, chemokines, and growth factors involved in the pathogenesis. Result(s): Our data indicated that not only were the levels of several of these host factors differentially modulated between the two study groups, but also that SARS-CoV-2 infected subjects presented with greater frequency of several important inflammatory cytokines and chemokines such as CCL2, CCL3, IFNG, entry receptors such as ACE2, TMRPS11A, and host restriction factors including LY6E and ZBP1. Conclusion(s): TaqMan array plates provide a fast, midthroughput solution to determine the levels of several virus and host-associated factors in various cell types and add to our understanding of how the pathogenesis may vary depending on gender, age, infection site etc.
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Objective To explore the potential common mechanism and active ingredients of Reduning Injection against SARS, MERS and COVID-19 through network pharmacology and molecular docking technology. Methods The TCMSP database was used to retrieve the chemical components and targets of Artemisiae Annuae Herba, Lonicerae Japonicae Flos and Gardeniae Fructus in Reduning Injection. The gene corresponding to the target was searched by UniProt database, and Cytoscape 3.8.2 was used to build a medicinal material-compound-target (gene) network. Three coronavirus-related targets were collected in the Gene Cards database with the key words of "SARS""MERS" and "COVID-19", and common target of three coronavirus infection diseases were screened out through Venny 2.1.0 database. The common targets of SARS, MERS and COVID-19 were intersected with the targets of Reduning Injection, and the common targets were selected as research targets. Protein-protein interaction (PPI) network map were constructed by Cytoscape3.8.2 software after importing the common targets into the STRING database to obtain data. R language was used to carry out GO biological function enrichment analysis and KEGG signaling pathway enrichment analysis, histograms and bubble charts were drew, and component-target-pathway network diagrams was constructed. The key compounds in the component-target-pathway network were selected for molecular docking with important target proteins, novel coronavirus (SARS-CoV-2) 3CL hydrolase, and angiotensin-converting enzyme II (ACE2). Results 31 active compounds and 207 corresponding targets were obtained from Reduning Injection. 2 453 SARS-related targets, 805 MERS-related targets, 2 571 COVID-19-related targets, and 786 targets for the three diseases. 11 common targets with Reduning Injection: HSPA5, CRP, MAPK1, HMOX1, TGFB1, HSP90AA1, TP53, DPP4, CXCL10, PLAT, PRKACA. GO function enrichment analysis revealed 995 biological processes (BP), 71 molecular functions (MF), and 31 cellular components (CC). KEGG pathway enrichment analysis screened 99 signal pathways (P < 0.05), mainly related to prostate cancer, fluid shear stress and atherosclerosis, hepatocellular carcinoma, proteoglycans in cancer, lipid and atherosclerosis, human T-cell leukemia virus 1 infection, MAPK signaling pathway, etc. The molecular docking results showed that the three core active flavonoids of quercetin, luteolin, and kaempferol in Reduning Injection had good affinity with key targets MAPK1, PRKACA, and HSP90AA1, and the combination of the three active compounds with SARS-CoV-2 3CL hydrolase and ACE2 was less than the recommended chemical drugs. Conclusion Reduning Injection has potential common effects on the three diseases of SARS, MERS and COVID-19. This effect may be related to those active compounds such as quercetin, luteolin, and kaempferol acting on targets such as MAPK1, PRKACA, HSP90AA1 to regulate multiple signal pathways and exert anti-virus, suppression of inflammatory storm, and regulation of immune function.Copyright © 2022 Drug Evaluation Research. All rights reserved.
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Background: High-titer neutralizing anti-cytokine autoantibodies have been shown to be involved in several acquired diseases, including pulmonary alveolar proteinosis, cryptococcal meningitis, and disseminated/extrapulmonary Nocardia infections (anti-GM-CSF autoantibodies), disseminated mycobacterial disease (anti-IFN-gamma autoantibodies), and some cases of severe COVID-19 infection (anti-type 1 interferons). Currently, patient blood samples are shipped via courier and require temperaturecontrolled conditions for transfer. This method is expensive and requires patients to have access to medical personnel to draw the blood. However, the well-established technique of collecting blood on a paper card as a dried blood spot (DBS) for diagnosis offers a point of care alternative which can be performed with a simple finger prick. This method is less invasive, cheaper, and allows for easy transport of patient samples. Method(s): 30 uL of whole blood from patients was blotted on filter paper and stored at 4C until use. The filter paper was hole punched and each punched spot was eluted with 150 uL of a 0.05% Tween PBS solution at room temperature overnight. The eluate was screened for anti-cytokine autoantibodies using a particle-based approach. Patient plasma was also screened in conjunction for comparison. Result(s): We confirmed the presence of autoantibodies in the DBS eluate from 4 previously diagnosed patients with anti-GM-CSF autoantibodies and 2 patients with anti-IFN-gamma autoantibodies. Functional studies showed the DBS eluate from a patient with anti-GM-CSF autoantibodies was able to block GM-CSF-induced STAT-5 phosphorylation in normal PBMC. As a proof of concept and to increase the number of patients evaluated, we also confirmed the presence of anti-cytokine autoantibodies using dried plasma eluate from 9 patients with known anti-GM-CSF autoantibodies and 9 patients with anti-IFN-gamma autoantibodies. Levels detected in DBS analyses were comparable to the levels found in plasma from the same patients not subjected to blotting and elution. Temperature studies showed that the autoantibodies were detected at similar levels when stored at 4C, 25C, and 40C for a week. Conclusion(s): The diagnosis of pathogenic anti-cytokine autoantibodies should be considered in the context of unusual or adult-onset infections, and screening for this diagnosis can be performed with dried blood spot testing.Copyright © 2023 Elsevier Inc.
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Neutrophilic granulocytes (NG) are the main drivers of pathological inflammation in COVID-19. Objective. To specify the mechanisms of immunopathogenesis of COVID-19 based on a comparative immunological study of the number and phenotype of CD16+SD62L+CD11b+CD63- and CD16+SD62L+CD11b+CD63+ subsets with an assessment of their effector functions against changing profile of NG-associated cytokines IL-8, IL-18, IL-17A, VEGF-A, IFNalpha, and IFNgamma. Patients and methods. In patients with moderate-to-severe and severe COVID-19, we determined IL-1beta, TNFalpha, IL-6, IL-8, IL-18, IL-17A, VEGF-A, IFNalpha, and IFNgamma (ELISA), the phenotype of CD16+SD62L+CD11b+CD63- and CD16+SD62L+CD11b+CD63+ subsets, NF-kappaB-NG (CYTOMICS FC500), phagocytically active NG (%), neutrophil extracellular traps (NETs), NG in apoptosis, and the activity of NADPH oxidase. Results. In COVID-19 against the background of IFNalpha and IFNgamma production blockade and high levels of NG-associated IL-8, IL-18, IL-17A, VEGF-A, a reduction in the number of mature and functionally active CD16brightSD62LbrightCD11bbrightCD63-NG subsets was revealed, as well as an increase in the number of CD16dimSD62LdimSD11bbrightCD63-NG subsets with an immunosuppressive phenotype and CD16brightSD62LbrightSD11bbrightCD63bright-NG subsets with high cytotoxic activity and ability to form NETs, a decrease in the percentage of phagocytically active NG and an increase in the activity of NADPH oxidase, NETs, and NG in apoptosis. Conclusion. IFNalpha deficiency provokes a hyperergic response of NG-associated cytokines, which leads to the formation of uncontrolled immune inflammation involving NG subsets with an immunosuppressive and cytotoxic phenotype, exacerbating the course of COVID-19. The use of recombinant IFNalpha-2b with antioxidants (Viferon) in the early stages of the disease can help to restore immune homeostasis, normalize the level of NG-associated cytokines, reduce NERTs, and achieve good clinical efficacy.Copyright © 2022, Dynasty Publishing House. All rights reserved.
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The current outbreak of COVID-19 is caused by the SARS-CoV-2 virus that has affected > 210 countries. Various steps are taken by different countries to tackle the current war-like health situation. In India, the Ministry of AYUSH released a self-care advisory for immunomodulation measures during the COVID-19 and this review article discusses the detailed scientific rationale associated with this advisory. Authors have spotted and presented in-depth insight of advisory in terms of immunomodulatory, antiviral, antibacterial, co-morbidity associated actions, and their probable mechanism of action. Immunomodulatory actions of advised herbs with no significant adverse drug reaction/toxicity strongly support the extension of advisory for COVID-19 prevention, prophylaxis, mitigations, and rehabilitation capacities. This advisory also emphasized Dhyana (meditation) and Yogasanas as a holistic approach in enhancing immunity, mental health, and quality of life. The present review may open-up new meadows for research and can provide better conceptual leads for future researches in immunomodulation, antiviral-development, psychoneuroimmunology, especially for COVID-19.Copyright © 2021, Institute of Korean Medicine, Kyung Hee University.
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Background/Objectives: COVID-19 still represents a lifethreatening disease in individuals with a specific genetic background. We successfully applied a new Machine Learning method on WES data to extract a set of coding variants relevant for COVID- 19 severity. We aim to identify personalized add-on therapy. Method(s): A subset of identified variants, "actionable" by repurposed drugs, were functionally tested by in vitro and in vivo experiments. Result(s): Males with either rare loss of function variants in the TLR7 gene or L412F polymorphism in the TLR3 gene benefit from IFN-gamma, which is specifically defective in activated PBMCs, restoring innate immunity. Females heterozygous for rare variants in the ADAMTS13 gene and males with D603N homozygous polymorphism in the SELP gene benefit from Caplacizumab, which reduces vWF aggregation and thrombus formation. Males with either the low-frequency gain of function variant T201M in CYP19A1 gene or with poly-Q repeats >=23 in the AR gene benefit from Letrozole, an aromatase inhibitor, which restores normal testosterone levels, reducing inflammation and which rescues male golden hamsters from severe COVID-19. Conclusion(s): By adding these commonly used drugs to standard of care of selected patients, the rate of intubation is expected to decrease consistently, especially in patients with high penetrance rare genetic markers, mitigating the effect of the pandemic with a significant impact on the healthcare system.
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Objective This study aimed to investigate the immune response of a pregnant woman who recovered from the coronavirus disease 2019 (COVID_RS) by using single-cell transcriptomic profiling of peripheral blood mononuclear cells (PBMCs) and to analyze the properties of different immune cell subsets. Methods PBMCs were collected from the COVID_RS patient at 28 weeks of gestation, before a cesarean section. The PBMCs were then analyzed using single-cell RNA sequencing. The transcriptional profiles of myeloid, T, and natural killer (NK) cell subsets were systematically analyzed and compared with those of healthy pregnant controls from a published single-cell RNA sequencing data set. Results We identified major cell types such as T cells, B cells, NK cells, and myeloid cells in the PBMCs of our COVID_RS patient. The increase of myeloid and B cells and decrease of T cells and NK cells in the PBMCs in this patient were quite distinct compared with that in the control subjects. After reclustering and Augur analysis, we found that CD16 monocytes and mucosal-Associated invariant T (MAIT) cells were mostly affected within different myeloid, T, and NK cell subtypes in our COVID_RS patient. The proportion of CD16 monocytes in the total myeloid population was increased, and the frequency of MAIT cells in the total T and NK cells was significantly decreased in the COVID-RS patient. We also observed significant enrichment of gene sets related to antigen processing and presentation, T-cell activation, T-cell differentiation, and tumor necrosis factor superfamily cytokine production in CD16 monocytes, and enrichment of gene sets related to antigen processing and presentation, response to type II interferon, and response to virus in MAIT cells. Conclusion Our study provides a single-cell resolution atlas of the immune gene expression patterns in PBMCs from a COVID_RS patient. Our findings suggest that CD16-positive monocytes and MAIT cells likely play crucial roles in the maternal immune response against severe acute respiratory syndrome coronavirus 2 infection. These results contribute to a better understanding of the maternal immune response to severe acute respiratory syndrome coronavirus 2 infection and may have implications for the development of effective treatments and preventive strategies for the coronavirus disease 2019 in pregnant women.Copyright © Wolters Kluwer Health, Inc. All rights reserved.
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Background & Aim: Immunocompromised patients are susceptible to high-risk opportunistic infections and malignant diseases. If available, most antiviral and antifungal drugs are quite toxic, relatively ineffective, and induce resistance in the long term. Methods, Results & Conclusion(s): We have previously demonstrated the safety of adoptive cell therapy for COVID-19 patients with CD45RA negative cells containing SARS-CoV-2-specific T cells from a donor, chosen based on HLA compatibility and cellular response to SARS-CoV-2 peptide pools. After finishing a Phase 2 randomized multicenter clinical trial (RELEASE, NCT04578210), we concluded that the infusion is safe, effective, accelerates lymphocyte recovery and shows hallmarks of an immune response. To use adoptive cell therapy to treat COVID-19 it would be necessary to develop a biobank of living drugs. For that, we examined the immune evolution performing a longitudinal analysis from previously SARS-CoV-2 infected and infection- naive individuals covering 21 months from infection. Cellular responses were maintained over time while humoral responses increased after vaccination but were gradually lost. Therefore, the best donors would be recovered individuals and two months after vaccination. We also evaluated the effect of dexamethasone (current standard of care treatment for COVID-19 and other infections involving lymphopenia) and Interleukin-15 (cytokine involved in T-cell maintenance and survival) on CD45RA negative. Dexamethasone did not alter cell functionality, proliferation or phenotype at a clinical-practice concentration, while interleukin-15 increased the memory T-cell and T-regulatory cell activation state, and interferon gamma release. Furthermore, we applied the adoptive passive transfer of CD45RA negative cells containing pathogen-specific memory T-cells to other infectious diseases characterized by sustained lymphopenia. We infused six immunocompromised patients with Cytomegalovirus, BK virus, Aspergillus, and Epstein-Barr virus lymphoproliferative disease. Patients experienced pathogen clearance, resolution of symptoms and lymphocyte increase. Transient microchimerism was detected in three patients. The use of CD45RA negative cells containing specific memory T cells of a third-party donor for treating severe pathogenic diseases in immunocompromised patients is feasible, safe, and effective, and has an advantage over other cell therapies such as lower costs and a less complex regulatory environment.Copyright © 2023 International Society for Cell & Gene Therapy
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Covid-19 is a viral disease that affects humans caused by a type of virus belonging to the family Coronaviridae called the SARS-CoV-2 virus. The parasitic infection associated with this disease affects the host's immune response regulation. The levels of IgG and IgM of Toxoplasma gondii in the serum of patients with COVID-19 were measured by immunoassay of the patient's sera by ELISA. Also, the level of interferon-gamma (IFN-gamma) in a covid-19 patient with or without Toxoplasmosis was evaluated. 120 samples were collected, 60 were positive for COVID-19, confirmed by clinically and radiographic examination, and 30 were in the control group. The results showed a significant difference between the infection with Covid-19 and T. gondii during the chronic phase of Toxoplasmosis compared to the negative relationship in the acute phase. The results of INF-gamma levels among Covid-19 patients were positive for all samples included in the test (30 Covid-19 patients and 30 patients COVID-19(+)/T. gondii IgG) compared to the control group. The chronic form of Toxoplasma disease, due to change in the production of this interferon, the COVID-19 infection has changed.Copyright © 2023 by Razi Vaccine & Serum Research Institute.
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While the exploration into biomolecules for diagnostic and prognostic devices continues to develop, many molecules continue to be examined for individual diseases or treatments. Consequently, it can be difficult to fully understand the scope of one individual molecule's current and potential clinical utilization. The scope of this study aimed to assess the potential of Interferon Gamma-induced Protein 10 (IP-10) as a biomarker in a wide variety of diseases, both as a main and supplemental indicator of disease infection and progression. IP-10 is a chemokine secreted in response to IFN-gamma playing a major role in the activation and regulation of inflammatory and immune responses within the body. Currently, IP-10 has displayed potential application in diseases such as COVID-19, tuberculosis, sepsis, Kawasaki disease, cancer, and many more. Molecular assays developed for the detection of IP-10 take longer testing time, sophisticated instrument utilization, and need more sample volumes. These cannot be utilized for bedside patient monitoring during the illness state of the patient. Biosensing tools are alternative methods used at clinical sites due to their rapid results. Though many types of sensing mechanisms established for the detection of disease biomarkers such as optical, piezoelectric sensors, and electrochemical biosensors are far beyond the other sensing methods due to their ease of mechanism, rapid results, and portable nature. IP-10 has been a promising biomarker in different diseases, evaluation of IP-10 levels at different time points of treatments is necessary. To achieve this, current conventional methods cannot be used and thus a portable device that provides rapid results is in demand. Such point-of-care (POC) device development for IP-10 analysis is very crucial in the current scenario. Beyond this, the clarification of its physiological role in healthy and infected individuals could allow for more proper utilization in clinical diagnoses, prognoses, treatment monitoring, and more. Overall, this study was developed to summarize the associations currently created between levels of IP-10 and other biomolecules and diseases.Copyright © 2023 The Author(s)
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Intro: VLA2001 is a highly-purified, inactivated whole-virus SARS-CoV-2 vaccine based on a dual-adjuvant system of Alum and CpG1018 for induction of a robust immune response. The vaccine was designed using a well-established technology platform and has received full marketing authorization in Europe. In a pivotal Phase 3 trial, VLA2001 demonstrated superior neutralizing antibody geometric mean titers (GMT) to the comparator, AstraZeneca's AZD1222, as well as non-inferior seroconversion rates two weeks after priming. The extension of the Phase 3 trial evaluated safety and immunogenicity of homologous and heterologous booster vaccinations of VLA2001. Method(s): This is a randomized observer-blind controlled, pivotal trial conducted in the UK in participants aged >=18 years who were randomly assigned 2:1 to receive two doses of VLA2001 or AZD1222, 28 days apart. A booster with VLA2001 was administered to eligible participants at 7 to 8 months after priming. The primary safety outcome was the frequency and severity of any adverse event following the booster vaccination. The primary immunogenicity outcomes were the GMT and fold increase of neutralizing antibodies against SARS-CoV-2 two weeks after the booster vaccination. The study is registered under NCT04864561. Finding(s): A booster dose of VLA2001 is well-tolerated in both AZD1222 and VLA2001 primed participants. High neutralizing antibody titers and fold- increases were generated two weeks following a booster of VLA2001. Cross- neutralizing serological responses against Delta and the Omicron BA.4/BA.5 variants of concern are elicited following a homologous or heterologous booster dose in VLA2001 or AZD1222 primed participants, respectively. Additionally, VLA2001 induced broad T-cell responses with antigen-specific IFN-gamma producing T-cells against the Spike, the Nucleocapsid and the Membrane protein. Conclusion(s): Homologous and heterologous booster doses of VLA2001 demonstrated a favorable tolerability profile irrespective of priming and induced broadly reactive neutralizing antibodies against the ancestral virus and variants of concern, including the currently circulating BA.4/BA.5.Copyright © 2023
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Background: Aim of the study was to analyze neutralizing activity against BA.5,BQ.1.1 and T cell response after 3rd booster dose [3BD (5th shot)] with BA.4/5 bivalent vaccine by hybrid immunity (HI) and CD4 count in advanced PLWH. Method(s): In PLWH with previous AIDS and/or CD4< 200/mm3 receiving 3BD (original strain/BA.4/5),immunogenicity was assessed at time of 3BD (T0) and at day 15 (T1) by microneutralization assay [MNA90] against Omicron BA.5, BQ.1.1 and by IFNgamma-ELISA. PLWH were stratified by HI vs. nHI and by CD4 count at T0 ( >or< 500/3). For crude mean comparisons, neutralizing antibodies (nAbs) were expressed in natural scale and fold changes, IFNgamma and all values for regression analyses in log2 scale, paired t-test used to test changes over T0-T1. Two 2-arms parallel trials were emulated: HI and CD4 count as exposure, log2 nAbs and IFNgamma as outcome. Average treatment effect (ATE) of the two exposures were estimated by marginal models weighted for potential confounders (age, CD4 nadir, years from AIDS;when HI was the exposure also CD4 count). Result(s): N=48 PLWH on ART, 15% female, median age 56 yrs, 45% >1 comorbidity, 87% with previous AIDS, median CD4 nadir 44 cell/mm3 (16-102), 98% with HIV-RNA < 50 cps/mL. A significant increase of nAbs against BA.5 (fold-increase 8.8,p< 0.0001) and BQ.1.1 (6.4, p< 0.0001) was observed from T0 to T1. At T1, in nHI (n=29), mean nAb was 176 and 53 against BA.5 and BQ.1.1, respectively, with a fold change reduction (FCR) vs BA.5 of 3.3;in HI (n=19), 496 and 128, respectively, with a FCR of 3.8 (Fig.1A). After controlling for confounders, HI was associated with a higher level of neutralizing response against BA.5 [ATE=1.17 log2 (95%CI 0.34;2.00), P=0.006] but not against BQ.1.1 [0.65 log2 (-0.18;1.48), p=0.124]. At T1, among PLWH with CD4 count< 500 (n=29), mean nAb was 290.8 and 83.9 against BA.5 and BQ.1.1, respectively, with a FCR of 3.4;in those with CD4 count >500 (n=19), 230.4 and 64.3, respectively, with a FCR of 3.6 (Fig. 1C).There was no impact of CD4 count on neutralization after controlling for potential confounding factors. No evidence for a difference between T0 and T1 was detected for IFNg (Fig.1B,D). Conclusion(s): In PLWH with advanced diseases, bivalent BA.5 3BD elicited strong neutralization against BA.5, and retained cross-neutralization against BQ.1.1, even if 3 times lower. HI but not CD4 count >500 appeared to enhance neutralization against BA.5. Importantly, bivalent vaccine appeared to have no effect on T-cell mediated response. (Figure Presented).
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Background: A significant portion of individuals experience persistent symptoms months after SARS-CoV-2 infection, broadly referred to as Long COVID (LC). Although the frequencies of subsets of SARS-CoV-2-specific T cells have been shown to differ in individuals with LC relative to those with complete recovery, a deep dive into phenotypic and functional features of total and SARSCoV- 2-specific T cells from individuals with LC has yet to be performed. Method(s): Here, we used CyTOF to characterize the phenotypes and effector functions of T cells from LIINC cohort. The median age was 46, the cohort was 55.8% female, and 9/43 had been hospitalized. Participants were reported a median of 7 LC symptoms at 8 months. SARS-CoV-2-specific total antibody levels were also measured in concurrent sera. Manual gating was used to define T cell subsets, SPICE analyses for polyfunctionality, T cell clustering for phenotypic features, and linear regression for correlation. Permutation tests, Student's t tests, and Welch's t test were used for statistical analysis. Result(s): SARS-CoV-2 total antibody responses were elevated in the LC group (p=0.043), and correlated with frequencies of SARS-CoV-2-specific T cells in those without LC (r=0.776, p< 0.001) but not those with LC. While the frequencies of total SARS-CoV-2-specific CD4+ and CD8+ T cells were similar between individuals with and without LC, those from individuals without LC tended to be more polyfunctional (co-expressing IFNgamma, TNFalpha, IL2, and/or MIP1beta). CD4+ T cells from individuals with LC harbored higher frequencies of Tcm (p=0.003), Tfh (p=0.037), and Treg subsets (p=0.0412), and preferentially expressed a variety of tissue homing receptors including CXCR4 and CXCR5 (p=0.037). SARS-CoV-2-specific CD4+ T cells producing IL6, albeit rare, were observed exclusively among those with LC (p=0.016). In addition, participants with LC harbored significantly higher frequencies of SARS-CoV-2-specific CD8+ T cells co-expressing exhaustion markers PD1 and CTLA4 (p=0.018). Conclusion(s): Long COVID is characterized by global phenotypic differences in the CD4+ T cell compartment in ways suggesting preferential migration of these cells to inflamed mucosal tissues. Individuals with LC also harbor higher numbers of exhausted SARS-CoV-2-specific CD8+ T cells, potentially implicating viral persistence. Finally, our data additionally suggest that individuals with LC may uniquely exhibit an uncoordinated T cell and antibody response during COVID-19 convalescence.
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Background: The pathogenetic mechanisms behind the development of long- COVID (LC) are largely unknown. Because both plasma SARS-CoV-2 RNAemia and dysregulated immunity have been correlated with COVID-19 severity, we evaluated whether they are associated with LC. Method(s): We consecutively enrolled unvaccinated hospitalized COVID-19 patients during acute-COVID-19 (T0) in March-October 2020 who either developed LC at a follow-up visit 2-3 months from virologic clearance (T1) or did not. LC was defined as persistence >=2 months after recovery of >=1 symptom: anosmia, dysgeusia, fever, gastrointestinal symptoms, dyspnoea, fatigue, musculoskeletal pain, muscle weakness, brain fog. We measured: SARS-CoV-2 RNAemia (RT-qPCR, log10(copies/mL)), magnitude (ELISA, AUC) and functionality (pseudovirus neutralization, ID50;Fc-mediated functions, %ADCC) of SARS-CoV-2-specific antibodies, SARS-CoV-2-specific B and CD4-T-cells (Immunophenotype, AIM and ICS assays). Result(s): We enrolled 48 COVID-19 individuals, 38/48 (79.2%) developed LC (LC+) and 10 did not (LC-). LC+ and LC- had similar co-morbidities and symptoms in the acute phase (Fig.1A), and the majority showed a radiologically documented SARS-CoV-2 pneumonia. The SARS-CoV-2 RNAemia did not differ between groups at both time points. The levels of RBD-specific Abs, as well as their functionality, appeared to increase over time in the LC- group but not in the LC+ (Fig.1B-D). Similarly, a trend towards increased RBD-specific B-cells was observed over time in the LC- group but not in LC+ (Fig.1E). B-cell immunophenotyping showed a significant increase over time of classical memory B cells (MBCs) at the expenses of activated MBCs (Fig.1F-G) as well as an IgA class-switching in the LC- group compared to LC+ (Fig.1H-I). Furthermore, LC+ showed a faster decline of SARS-CoV-2-specific (CD69+CD137+) CD4- TEMRA and CD4-TEM (Fig.1L-M). Finally, IFN-gamma-producing TREG of LC- individuals increased over time (Fig.1N). Conclusion(s): Acutely ill, hospitalized COVID-19 patients developing LC feature a dysregulated SARS-CoV-2-specific humoral as well as B- and T-cell response, in both magnitude and functionality, suggesting a link between dysregulated SARS-CoV-2-specific adaptive immunity and LC development. The fine understanding of the factors contributing to such dysregulation in LC patients is strongly needed, that might further inform targeted therapeutic interventions. (Figure Presented).
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Respiratory viral infections are important cause of morbidity and mortality in early life. The relative influence of host and viral factors possibly contribute to the disease pathogenesis. Predisposing conditions like prematurity, Low birth weight and congenital heart diseases etc. have been incriminated in the disease progression. The development of cough, wheezing, and tachypnea, usually peaking on days 4 to 5, go parallel with host cytokine responses and viral load. Various host cytokines, chemokines and molecules involved in the immune response against RSV infection might be responsible for the outcome of the disease process. Nasopharyngeal aspirates (NPAs) from children (n = 349) between 2013-2017 were subjected for IL-17A, IFN-gamma, TNF-alpha, IL-10, IL-6 levels by CBA and MMP-9 and TIMP-1 levels by ELISA. The viral load in RSV positive samples and cytokine levels were correlated with the WHO criteria for acute lower respiratory tract illness (ALRTI). RSV viral load, Pro-inflammatory cytokine (TNF-alpha) levels in severe ALRTI patients were significantly higher than the ALRTI patients [p<0.001]. Whereas Th17 cytokine (IL-17) was found to be significantly higher (p<0.05) in ALRTI patients than severe patients. MMP-9 is secreted in higher levels in severe ALRTI patients (n = 77) in comparison to Acute LRTI patients (n = 35) with an increase of thirty seven fold (p<0.001). Thus, the study highlights the role of TNF -alpha, IL-17 and Th2 cytokine biasness in the pathogenesis of RSV disease with the possible contribution of higher MMP-9/TIMP-1 ratio as a bad prognostic marker towards disease severity. To study the gene expression of autophagy and mTOR signalling pathways in RSV infected children with ALRTI. Nasopharyngeal aspirate (NPA) samples (n = 145) from children suffering from ALRTI were subjected for detection of RSV (Oct 2019 to March 2020). Semi-quantitative gene expression analysis for 5 representative genes each of mTOR signalling and autophagy pathway were performed in respiratory tract epithelial cells using 25 RSV positive cases and 10 healthy controls subjects. Autophagy gene expression analysis revealed significant upregulation in NPC1 and ATG3 autophagy genes. mTOR, AKT1 and TSC1 genes of mTOR pathway were significantly down-regulated in RSV positive patients except RICTOR gene which was significantly upregulated. Thus, survival of RSV within autophagosome might have been facilitated by upregulation of autophagy and downregulation of mTOR signalling genes. To assess the impact of SARS-CoV2 pandemic on RSV, samples were collected from children with ALRTIs admitted to emergency, PICU and indoor admissions during pre-pandemic period (October 2019 to February 2020;n = 166) and during COVID-19 Pandemic (July 2021 to July 2022;n = 189, SARS-CoV2 negative). These NP swabs were analyzed for pdm InfA H1N1, InfA H3N2, Inf B, RSV, hMPV, hBoV, hRV, PIV-2 and PIV-3 by PCR. Higher proportion of children with ALRTIs have had virus/es isolated during pre-pandemic period than during pandemic period (p<0.001). During pre-pandemic period, significantly higher proportion of children had RSV positivity (p<0.001);and significantly lower positivity for hRV (p<0.05), hMPV (p<0.05), and hBoV (p <= 0.005). The occurrence of COVID-19 pandemic has significantly impacted the frequency and pattern of detection of RSV among hospitalized children with LRTIs. RSV Fusion protein plays a critical role in the entry of the virus into the host cell by initiating the fusion of host and viral membranes. It happens to be a target of neutralizing antibodies paving the way as a vaccine candidate. Hence effort was made to introduce point mutation in hRSV fusion protein which can confer stability in its prefusion form. In-silico a stable structure of RSV fusion protein was generated making it a potential vaccine candidate. The timely diagnosis of RSV infection in this population is important for initiating therapy and instituting appropriate infection prevention measures. Serological testing is not widely used for the diagnosis of RSV. C ll Cultures including shell vial culture were used for RSV diagnosis. However, culture approaches lack sensitivity, often quite significantly, compared to nucleic acid amplification assays for the diagnosis of RSV infections. Molecular multiplex assays now offer increased sensitivity for a more accurate diagnosis. However issues with the use of these types of commercial panel assays include the requirement for substantial training, quality systems, and infrastructure to maintain and run these assays and many a times identification of viruses where the true pathogenic potential of those multiple viruses are debatable. Studies are available with laboratory- developed nucleic acid amplification test systems for the detection of RSVA and RSVB in clinical specimens either by PCRbased technologies or RT-LAMP. Gene targets of laboratory-developed molecular assays point towards M gene and the N gene in RSVA and -B with the benefits of flexibility to modify assays when targets are under evolutionary pressure to change, as well as a perceived initial low cost to carry out testing.
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Background: Resident memory T cells (TRM) present at the respiratory tract may be essential to enhance early SARS-CoV-2 viral clearance, thus limiting viral infection and disease. While long-term antigen-specific TRM are detectable beyond 11 months in the lung of convalescent COVID-19 patients after mild and severe infection, it is unknown if mRNA vaccination encoding for the SARS-CoV-2 S-protein can induce this frontline protection. Method(s): We obtained cross-sectional paired blood and lung biopsy samples from patients (n=30) undergoing lung resection for various reasons and assigned them to one of four groups: I.) uninfected unvaccinated individuals (n=5), II.) unvaccinated long-term SARS-CoV-2 convalescent individuals (between 6.0-10.5 months post-infection;n=9), III.) uninfected and long-term vaccinated individuals (between 6.0-7.7 months after the second or third dose;n=10), and IV.) uninfected and short-term vaccinated individuals (between 1.3-1.8 months after the third or fourth dose;n=6). We determined the presence of SARS-CoV-2-specific CD4+ and CD8+ T cells in blood and lung samples after exposure of cells to M, N, and S peptide pools, followed by flow cytometry to detect TRM cells expressing interferon (IFN)gamma and/or CD107a, as a degranulation marker. Result(s): We found that the frequency of CD4+ T cells secreting IFNgamma in response to S-peptides was variable but detectable in blood and lung up to 8 months after mRNA vaccination. Moreover, the IFNgamma response of CD4+ T cells in the lung of mRNA-vaccinated patients was similar to the response found in convalescent patients. However, in mRNA-vaccinated patients, lung responses presented less frequently with a TRM phenotype compared to convalescent infected individuals and, strikingly, polyfunctional CD107a+ IFNgamma+ TRM were virtually absent in vaccinated patients. Conclusion(s): mRNA vaccines might induce memory responses within the lung parenchyma in some patients, potentially contributing to the overall disease control. However, the robust and broad TRM response established in convalescent-infected individuals may offer advantages at limiting disease if the virus is not blocked by initial mechanisms of protection, such as neutralization. Our results warrant investigation of mucosal vaccine-induced resident T cell responses in establishing superior site-specific protective immunity.
ABSTRACT
Background: Vaccination plays a major role in controlling SARS-CoV2 infection but faces the issue of short-term protection. Beyond the generation of Abs, induction of memory CD8+T cells with stem cell-like (Tscm) properties is essential for long-term immunity to viruses. We have designed a sub-unit CD40.CoV-2 vaccine which targets Spike (S) and nucleocapsid (N) regions from SARS-CoV-2 to antigen presenting cells with comparable immunogenicity and protective effect than mRNA BNT162b2 (Pfizer-BioNTech) in preclinical models (Coleon S. EBioMed 2022). We hypothesized that CD40.CoV2 vaccine will elicit CD8+ Tscm cells. Method(s): CD40.CoV2 vaccine is a fully humanized mAb fused to RBD (aa 318-541) and N (aa 276-411). Humanized (hu) NSG mice (HIS-mice) (n=6/ group) received: i) CD40.CoV2 (10 mug equal to 1.3 mug of RBD, i.p.) +/- poly-ICLC (TLR3 agonist;50mug) or ii) BNT162b2 (1mug, i.m.);iii) IgG4.CoV2 (10mug, i.p.) as non-CD40-targeting control. Phenotype and function of splenic S and N-specific T cells were assessed at W5. Result(s): The CD40.CoV2 vaccine +/- poly-CLC induced significant S and N-specific Th1 huCD4+, cytokines-secreting huCD8+ T cells and RBD-specific IgG-switched huB cells as compared to mock injections and non-targeted IgG4.CoV2. CD40.CoV-2 vaccine +/- adjuvant induced higher frequencies of huCD8+ Tscm (CD95+ CD45RA+ CD62L+ ;median, (IQR) 22.4% (12.3-27.4) and 23% (20.7-29.1) +/- adjuvant, respectively) and central memory (TCM;CD45RA- CD62L+) CD8+ T cells (2.7% (2.3-6.2) and 5.1% (3.8-7.8) +/- adjuvant, respectively). In contrast, BNT162b2 induced predominantly effector memory (TEM, CD45RA- CD62L- ;median, (IQR) 63.1% (47.3-72.3)) but not Tscm (1.6% (0.9-6.6)) (figure). CD40.CoV-2 induced huCD8+ Tscm cells exhibit ;i) a higher proliferation index than TCM and TEM;ii) a functional profile secreting TNF and IFNgamma after restimulation with RBD or N peptides;cardinal features of Tscm cells. Conclusion(s): The CD40.SARS.CoV2, but not BNT162b2 vaccine, stimulates selective enrichment in S-and N-specific CD8+ Tscm cells that support longlasting anti-viral immunity. CD40.CoV2 sub-unit is under clinical development as a booster vaccine aimed to maintain durable anti-viral T and humoral responses. (Figure Presented).
ABSTRACT
Neutrophilic granulocytes (NG) are the main drivers of pathological inflammation in COVID-19. Objective. To specify the mechanisms of immunopathogenesis of COVID-19 based on a comparative immunological study of the number and phenotype of CD16+SD62L+CD11b+CD63- and CD16+SD62L+CD11b+CD63+ subsets with an assessment of their effector functions against changing profile of NG-associated cytokines IL-8, IL-18, IL-17A, VEGF-A, IFNalpha, and IFNgamma. Patients and methods. In patients with moderate-to-severe and severe COVID-19, we determined IL-1beta, TNFalpha, IL-6, IL-8, IL-18, IL-17A, VEGF-A, IFNalpha, and IFNgamma (ELISA), the phenotype of CD16+SD62L+CD11b+CD63- and CD16+SD62L+CD11b+CD63+ subsets, NF-kappaB-NG (CYTOMICS FC500), phagocytically active NG (%), neutrophil extracellular traps (NETs), NG in apoptosis, and the activity of NADPH oxidase. Results. In COVID-19 against the background of IFNalpha and IFNgamma production blockade and high levels of NG-associated IL-8, IL-18, IL-17A, VEGF-A, a reduction in the number of mature and functionally active CD16brightSD62LbrightCD11bbrightCD63-NG subsets was revealed, as well as an increase in the number of CD16dimSD62LdimSD11bbrightCD63-NG subsets with an immunosuppressive phenotype and CD16brightSD62LbrightSD11bbrightCD63bright-NG subsets with high cytotoxic activity and ability to form NETs, a decrease in the percentage of phagocytically active NG and an increase in the activity of NADPH oxidase, NETs, and NG in apoptosis. Conclusion. IFNalpha deficiency provokes a hyperergic response of NG-associated cytokines, which leads to the formation of uncontrolled immune inflammation involving NG subsets with an immunosuppressive and cytotoxic phenotype, exacerbating the course of COVID-19. The use of recombinant IFNalpha-2b with antioxidants (Viferon) in the early stages of the disease can help to restore immune homeostasis, normalize the level of NG-associated cytokines, reduce NERTs, and achieve good clinical efficacy.Copyright © 2022, Dynasty Publishing House. All rights reserved.
ABSTRACT
Background: People with HIV (PWH) older than age 55 have an enhanced risk of complications from SARS-CoV-2 infection. It is unclear whether COVID-19 vaccines with a booster are as durable in terms of immunogenicity in this cohort or whether these vaccines can destabilize HIV reservoirs. Method(s): We prospectively studied 91 PWH on cART aged 55 or over (n=91) and 23 age-matched individuals without HIV (control group, CG) who received three doses of COVID-19 vaccines (D1-D3) over 48 weeks. Participants received combinations of BNT162b2, mRNA-1273, and ChAdOx1. Of PWH, 42 were immune responders (IR), 20 were non-responders (INR), and 3 had a low-level viremia (LLV). Total and neutralizing Abs to SARS-CoV-2 spike (S) and RBD in sera and saliva, frequency of anti-RBD/NTD memory B cells (spectral flow cytometry), S-specific T cell immunity (IFN-g, IL-2 ELISpot) and HIV reservoirs in peripheral CD4+ T cells (IPDA) were measured. Result(s): No significant differences in vaccine regimens or dosing intervals were observed between PWH and CG. Vaccines elicited equally strong anti-S IgG in PWH vs CG in serum and saliva, and RBD IgG in serum. Serum Abs peaked at 4w after D3. Week 48 serum IgG in PWH vs CG were 916 vs 919 BAU/ mL for S (p=0.624) and 706 vs 752 for RBD (p=0.198), respectively. Week 48 median saliva S IgG: 48.1% AUC of the positive control in PWH vs 95.9% for CG (p=0.384). S IgA: 3.83 vs 20.5 in PWH vs CG (p=0.039). Median neutralizing titers post-D2 were significantly lower in PWH than in CG (NT50 82.9 vs 535, p< 0.001). However, after D3, at 48w, PWH had similar titers as CG: 309 vs 269 (p=0.745), mirroring an increase in RBD/NTD-specific B cells in PWH. Anti-S T cell cytokine responses were stronger in IR PWH after D2 and D3 than in CG. Week 48 S IL-2 responses: median 135 SFC/106 PBMC vs 43.8 (p< 0.001), but only 12.5 in INR (p=0.001 vs IR). COVID-19 vaccines did not affect the size of HIV reservoir in PWH (change in median frequency of intact proviruses from baseline: 95.0 vs 90.9, p=0.952), except in three LLV PWH (mean increase 93.7% at 48w). Conclusion(s): PWH aged 55 and over show diminished neutralizing Ab responses to SARS-CoV-2 with two vaccine doses which are 'rescued' after a booster. PWH have lower S-specific IgA in saliva after vaccination which may affect protection. Enhanced S-specific T cell immunity in PWH suggests Th1 imprinting from preexistent HIV infection. COVID-19 vaccines did not destabilize the HIV reservoir in most PWH but may pose potential risk in unsuppressed viremia.
ABSTRACT
Introduction/Aim: The spike protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus enables it to recognise and bind host receptors. These dynamics have been modelled in various cell types and immortalised lines, but rarely in primary airway epithelial cells (AEC), and especially not in children. Therefore, this study on AEC recapitulated earlier work testing the hypothesis that exposure to the spike protein would induce airway immune responses in airway cells of young children. Method(s): Primary AEC monolayer cultures from healthy children (n = 5, <10 years old, males = 5) were exposed to the spike protein S1 subunit (0.01, 1, and 10 mug/mL) over 48 h. Induced inflammatory cytokines, interleukin (IL) 6 and IL8, and viral-associated chemokines, CCL5 and CXCL10 were measured via ELISA. Basal receptor gene expression (ACE2 and TMPRSS2) was measured in monolayer (n = 5) and terminally differentiated (air-liquid interface [ALI];n = 5) cell models as well as in ex-vivo cells obtained directly from nasal brushings (n = 71). Generalised linear modelling, accounting for individual variability, identified any statistical difference (p < 0.05). Result(s): Exposure to the spike protein resulted in no increase in IL6 and IL8 production, however a significant (p < 0.05) decrease was observed at the highest dose tested (10 mug/mL). CXCL10 was only significantly induced at the highest dose (10 mug/mL) whereas CCL5 was not induced. When compared to ex-vivo samples, baseline expression of ACE2 and TMPRSS2 was significantly lower in monolayer cultures (~57- and ~4- fold respectively, p < 0.05), whereas ALI cultures had similar expression levels. Conclusion(s): The use of recombinant spike protein and monolayer cultures appears to not accurately model SARS-CoV-2 spike protein-host interactions. The lack of inflammatory responses may be attributed to the lower receptor gene expression in monolayer cultures. Future studies should utilise live virus and ALI cultures as a more biologically relevant model to study virus-host interactions.