Evaluation of the immunomodulatory effects of interleukin-10 on peripheral blood immune cells of COVID-19 patients: Implication for COVID-19 therapy.

Objective: Several therapies with immune-modulatory functions have been proposed to reduce the overwhelmed inflammation associated with COVID-19. Here we investigated the impact of IL-10 in COVID-19, through the ex-vivo assessment of the effects of exogenous IL-10 on SARS-CoV-2-specific-response using a whole-blood platform. Methods: Two cohorts were evaluated: in "study population A", plasma levels of 27 immune factors were measured by a multiplex (Luminex) assay in 39 hospitalized "COVID-19 patients" and 29 "NO COVID-19 controls" all unvaccinated. In "study population B", 29 COVID-19 patients and 30 NO COVID-19-Vaccinated Controls (NO COVID-19-VCs) were prospectively enrolled for the IL-10 study. Whole-blood was stimulated overnight with SARS-COV-2 antigens and then treated with IL-10. Plasma was collected and used for ELISA and multiplex assay. In parallel, whole-blood was stimulated and used for flow cytometry analysis. Results: Baseline levels of several immune factors, including IL-10, were significantly elevated in COVID-19 patients compared with NO COVID-19 subjects in "study population A". Among them, IL-2, FGF, IFN-γ, and MCP-1 reached their highest levels within the second week of infection and then decreased. To note that, MCP-1 levels remained significantly elevated compared with controls. IL-10, GM-CSF, and IL-6 increased later and showed an increasing trend over time. Moreover, exogenous addition of IL-10 significantly downregulated IFN-γ response and several other immune factors in both COVID-19 patients and NO COVID-19-VCs evaluated by ELISA and a multiplex analysis (Luminex) in "study population B". Importantly, IL-10 did not affect cell survival, but decreased the frequencies of T-cells producing IFN-γ, TNF-α, and IL-2 (p<0.05) and down-modulated HLA-DR expression on CD8+ and NK cells. Conclusion: This study provides important insights into immune modulating effects of IL-10 in COVID-19 and may provide valuable information regarding the further in vivo investigations.

Host biomarker-based quantitative rapid tests for detection and treatment monitoring of tuberculosis and COVID-19.

Diagnostic services for tuberculosis (TB) are not sufficiently accessible in low-resource settings, where most cases occur, which was aggravated by the COVID-19 pandemic. Early diagnosis of pulmonary TB can reduce transmission. Current TB-diagnostics rely on detection of Mycobacterium tuberculosis (Mtb) in sputum requiring costly, time-consuming methods, and trained staff. In this study, quantitative lateral flow (LF) assays were used to measure levels of seven host proteins in sera from pre-COVID-19 TB patients diagnosed in Europe and latently Mtb-infected individuals (LTBI), and from COVID-19 patients and healthy controls. Analysis of host proteins showed significantly lower levels in LTBI versus TB (AUC:0 · 94) and discriminated healthy individuals from COVID-19 patients (0 · 99) and severe COVID-19 from TB. Importantly, these host proteins allowed treatment monitoring of both respiratory diseases. This study demonstrates the potential of non-sputum LF assays as adjunct diagnostics and treatment monitoring for COVID-19 and TB based on quantitative detection of multiple host biomarkers.

Booster dose of SARS-CoV-2 messenger RNA vaccines strengthens the specific immune response of patients with rheumatoid arthritis: A prospective multicenter longitudinal study.

OBJECTIVES: To characterize the kinetics of humoral and T-cell responses in rheumatoid arthritis (RA)-patients followed up to 4-6 weeks (T3) after the SARS-CoV-2 vaccine booster dose. METHODS: Health care workers (HCWs, n = 38) and patients with RA (n = 52) completing the messenger RNA vaccination schedule were enrolled at T3. In each cohort, 25 subjects were sampled after 5 weeks (T1) and 6 months (T2) from the first vaccine dose. The humoral response was assessed by measuring anti-receptor-binding domain (RBD) and neutralizing antibodies, the T-cell response by interferon-γ-release assay (IGRA), T cell cytokine production, and B cell phenotype at T3 by flow cytometry. RESULTS: Patients with RA showed a significant reduction of antibody titers from T1 to T2 and a significant increase at T3. T-cell response by IGRA persisted over time in patients with RA, whereas it increased in HCWs. Most patients with RA scored positive for anti-RBD, neutralizing antibody and T-cell responses, although the magnitude was lower than HCWs. The spike-specific-cytokine response was mainly clusters of differentiation (CD)4+ T cells restricted in both cohorts and significantly lower with reduced interleukin-2 response and CD4-antigen-responding naïve T cells in patients with RA. Unswitched memory B cells were reduced in patients with RA compared with HCWs independently of vaccination. CONCLUSION: COVID-19 vaccine booster strengthens the humoral immunity in patients with RA even with a reduced cytokine response.

Evaluation of the immunomodulatory effects of interleukin-10 on peripheral blood immune cells of COVID-19 patients: Implication for COVID-19 therapy

Objective Several therapies with immune-modulatory functions have been proposed to reduce the overwhelmed inflammation associated with COVID-19. Here we investigated the impact of IL-10 in COVID-19, through the ex-vivo assessment of the effects of exogenous IL-10 on SARS-CoV-2-specific-response using a whole-blood platform. Methods Two cohorts were evaluated: in “study population A”, plasma levels of 27 immune factors were measured by a multiplex (Luminex) assay in 39 hospitalized “COVID-19 patients” and 29 “NO COVID-19 controls” all unvaccinated. In “study population B”, 29 COVID-19 patients and 30 NO COVID-19-Vaccinated Controls (NO COVID-19-VCs) were prospectively enrolled for the IL-10 study. Whole-blood was stimulated overnight with SARS-COV-2 antigens and then treated with IL-10. Plasma was collected and used for ELISA and multiplex assay. In parallel, whole-blood was stimulated and used for flow cytometry analysis. Results Baseline levels of several immune factors, including IL-10, were significantly elevated in COVID-19 patients compared with NO COVID-19 subjects in “study population A”. Among them, IL-2, FGF, IFN-γ, and MCP-1 reached their highest levels within the second week of infection and then decreased. To note that, MCP-1 levels remained significantly elevated compared with controls. IL-10, GM-CSF, and IL-6 increased later and showed an increasing trend over time. Moreover, exogenous addition of IL-10 significantly downregulated IFN-γ response and several other immune factors in both COVID-19 patients and NO COVID-19-VCs evaluated by ELISA and a multiplex analysis (Luminex) in “study population B”. Importantly, IL-10 did not affect cell survival, but decreased the frequencies of T-cells producing IFN-γ, TNF-α, and IL-2 (p<0.05) and down-modulated HLA-DR expression on CD8+ and NK cells. Conclusion This study provides important insights into immune modulating effects of IL-10 in COVID-19 and may provide valuable information regarding the further in vivo investigations.

Coinfection of tuberculosis and COVID-19 limits the ability to in vitro respond to SARS-CoV-2.

OBJECTIVES: The interaction of COVID-19 and tuberculosis (TB) are still poor characterized. Here we evaluated the immune response specific for Micobacterium tuberculosis (Mtb) and SARS-CoV-2 using a whole-blood-based assay-platform in COVID-19 patients either with TB or latent TB infection (LTBI). METHODS: We evaluated IFN-γ level in plasma from whole-blood stimulated with Mtb antigens in the Quantiferon-Plus format or with peptides derived from SARS-CoV-2 spike protein, Wuhan-Hu-1 isolate (CD4-S). RESULTS: We consecutively enrolled 63 COVID-19, 10 TB-COVID-19 and 11 LTBI-COVID-19 patients. IFN-γ response to Mtb-antigens was significantly associated to TB status and therefore it was higher in TB-COVID-19 and LTBI-COVID-19 patients compared to COVID-19 patients (p ≤ 0.0007). Positive responses against CD4-S were found in 35/63 COVID-19 patients, 7/11 LTBI-COVID-19 and only 2/10 TB-COVID-19 patients. Interestingly, the responders in the TB-COVID-19 group were less compared to COVID-19 and LTBI-COVID-19 groups (p = 0.037 and 0.044, respectively). Moreover, TB-COVID-19 patients showed the lowest quantitative IFN-γ response to CD4-S compared to COVID-19-patients (p = 0.0336) and LTBI-COVID-19 patients (p = 0.0178). CONCLUSIONS: Our data demonstrate that COVID-19 patients either TB or LTBI have a low ability to build an immune response to SARS-CoV-2 while retaining the ability to respond to Mtb-specific antigens.

In Vitro Models for Studying Entry, Tissue Tropism, and Therapeutic Approaches of Highly Pathogenic Coronaviruses.

Coronaviruses (CoVs) are enveloped nonsegmented positive-sense RNA viruses belonging to the family Coronaviridae that contain the largest genome among RNA viruses. Their genome encodes 4 major structural proteins, and among them, the Spike (S) protein plays a crucial role in determining the viral tropism. It mediates viral attachment to the host cell, fusion to the membranes, and cell entry using cellular proteases as activators. Several in vitro models have been developed to study the CoVs entry, pathogenesis, and possible therapeutic approaches. This article is aimed at summarizing the current knowledge about the use of relevant methodologies and cell lines permissive for CoV life cycle studies. The synthesis of this information can be useful for setting up specific experimental procedures. We also discuss different strategies for inhibiting the binding of the S protein to the cell receptors and the fusion process which may offer opportunities for therapeutic intervention.

Exploratory analysis to identify the best antigen and the best immune biomarkers to study SARS-CoV-2 infection.

BACKGROUND: Recent studies proposed the whole-blood based IFN-γ-release assay to study the antigen-specific SARS-CoV-2 response. Since the early prediction of disease progression could help to assess the optimal treatment strategies, an integrated knowledge of T-cell and antibody response lays the foundation to develop biomarkers monitoring the COVID-19. Whole-blood-platform tests based on the immune response detection to SARS-CoV2 peptides is a new approach to discriminate COVID-19-patients from uninfected-individuals and to evaluate the immunogenicity of vaccine candidates, monitoring the immune response in vaccine trial and supporting the serological diagnostics results. Here, we aimed to identify in the whole-blood-platform the best immunogenic viral antigen and the best immune biomarker to identify COVID-19-patients. METHODS: Whole-blood was overnight-stimulated with SARS-CoV-2 peptide pools of nucleoprotein-(NP) Membrane-, ORF3a- and Spike-protein. We evaluated: IL-1ß, IL-1Ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12p70, IL-13, IL- 15, IL-17A, eotaxin, FGF, G-CSF, GM-CSF, IFN-γ, IP-10, MCP-1, MIP-1α, MIP-1ß, PDGF, RANTES, TNF-α, VEGF. By a sparse partial least squares discriminant analysis we identified the most important soluble factors discriminating COVID-19- from NO-COVID-19-individuals. RESULTS: We identified a COVID-19 signature based on six immune factors: IFN-γ, IP-10 and IL-2 induced by Spike; RANTES and IP-10 induced by NP and IL-2 induced by ORF3a. We demonstrated that the test based on IP-10 induced by Spike had the highest AUC (0.85, p < 0.0001) and that the clinical characteristics of the COVID-19-patients did not affect IP-10 production. Finally, we validated the use of IP-10 as biomarker for SARS-CoV2 infection in two additional COVID-19-patients cohorts. CONCLUSIONS: We set-up a whole-blood assay identifying the best antigen to induce a T-cell response and the best biomarkers for SARS-CoV-2 infection evaluating patients with acute COVID-19 and recovered patients. We focused on IP-10, already described as a potential biomarker for other infectious disease such as tuberculosis and HCV. An additional application of this test is the evaluation of immune response in SARS-CoV-2 vaccine trials: the IP-10 detection may define the immunogenicity of a Spike-based vaccine, whereas the immune response to the virus may be evaluated detecting other soluble factors induced by other viral-antigens.

Spike is the most recognized antigen in the whole-blood platform in both acute and convalescent COVID-19 patients.

OBJECTIVES: To identify the best experimental approach to detect a SARS-CoV-2-specific T cell response using a whole-blood platform. METHODS: Whole-blood from 56 COVID-19 and 23 "NO-COVID-19" individuals were stimulated overnight with different concentrations (0.1 or 1 µg/mL) of SARS-CoV-2 PepTivator® Peptide Pools, including spike (pool S), nucleocapsid (pool N), membrane (pool M), and a MegaPool (MP) of these three peptide pools. ELISA was used to analyse interferon (IFN)-γ levels. RESULTS: The IFN-γ-response to every SARS-CoV-2 peptide pool was significantly increased in COVID-19 patients compared with NO-COVID-19 individuals. Pool S and MegaPool were the most potent immunogenic stimuli (median: 0.51, IQR: 0.14-2.17; and median: 1.18, IQR: 0.27-4.72, respectively) compared with pools N and M (median: 0.22, IQR: 0.032-1.26; and median: 0.22, IQR: 0.01-0.71, respectively). The whole-blood test based on pool S and MegaPool showed a good sensitivity of 77% and a high specificity of 96%. The IFN-γ-response was mediated by both CD4+ and CD8+ T cells, and independently detected of clinical parameters in both hospitalized and recovered patients. CONCLUSIONS: This easy-to-use assay for detecting SARS-CoV-2-specific T cell responses may be implemented in clinical laboratories as a powerful diagnostic tool.

In-vitro evaluation of the immunomodulatory effects of Baricitinib: Implication for COVID-19 therapy.

OBJECTIVE: Baricitinib seems a promising therapy for COVID-19. To fully-investigate its effects, we in-vitro evaluated the impact of baricitinib on the SARS-CoV-2-specific-response using the whole-blood platform. METHODS: We evaluated baricitinib effect on the IFN-γ-release and on a panel of soluble factors by multiplex-technology after stimulating whole-blood from 39 COVID-19 patients with SARS-CoV-2 antigens. Staphylococcal Enterotoxin B (SEB) antigen was used as a positive control. RESULTS: In-vitro exogenous addition of baricitinib significantly decreased IFN-γ response to spike- (median: 0.21, IQR: 0.01-1; spike+baricitinib 1000 nM median: 0.05, IQR: 0-0.18; p < 0.0001) and to the remainder-antigens (median: 0.08 IQR: 0-0.55; remainder-antigens+baricitinib 1000 nM median: 0.03, IQR: 0-0.14; p = 0.0013). Moreover, baricitinib significantly decreased SEB-induced response (median: 12.52, IQR: 9.7-15.2; SEB+baricitinib 1000 nM median: 8, IQR: 1.44-12.16; p < 0.0001). Baricitinib did modulate other soluble factors besides IFN-γ, significantly decreasing the spike-specific-response mediated by IL-17, IL-1ß, IL-6, TNF-α, IL-4, IL-13, IL-1ra, IL-10, GM-CSF, FGF, IP-10, MCP-1, MIP-1ß (p ≤ 0.0156). The baricitinib-decreased SARS-CoV-2-specific-response was observed mainly in mild/moderate COVID-19 and in those with lymphocyte count ≥1 × 103/µl. CONCLUSIONS: Exogenous addition of baricitinib decreases the in-vitro SARS-CoV-2-specific response in COVID-19 patients using a whole-blood platform. These results are the first to show the effects of this therapy on the immune-specific viral response.

Epidemic and pandemic viral infections: impact on tuberculosis and the lung: A consensus by the World Association for Infectious Diseases and Immunological Disorders (WAidid), Global Tuberculosis Network (GTN), and members of the European Society of Clinical Microbiology and Infectious Diseases Study Group for Mycobacterial Infections (ESGMYC).

Major epidemics, including some that qualify as pandemics, such as severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), HIV, influenza A (H1N1)pdm/09 and most recently COVID-19, affect the lung. Tuberculosis (TB) remains the top infectious disease killer, but apart from syndemic TB/HIV little is known regarding the interaction of viral epidemics and pandemics with TB. The aim of this consensus-based document is to describe the effects of viral infections resulting in epidemics and pandemics that affect the lung (MERS, SARS, HIV, influenza A (H1N1)pdm/09 and COVID-19) and their interactions with TB. A search of the scientific literature was performed. A writing committee of international experts including the European Centre for Disease Prevention and Control Public Health Emergency (ECDC PHE) team, the World Association for Infectious Diseases and Immunological Disorders (WAidid), the Global Tuberculosis Network (GTN), and members of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Study Group for Mycobacterial Infections (ESGMYC) was established. Consensus was achieved after multiple rounds of revisions between the writing committee and a larger expert group. A Delphi process involving the core group of authors (excluding the ECDC PHE team) identified the areas requiring review/consensus, followed by a second round to refine the definitive consensus elements. The epidemiology and immunology of these viral infections and their interactions with TB are discussed with implications for diagnosis, treatment and prevention of airborne infections (infection control, viral containment and workplace safety). This consensus document represents a rapid and comprehensive summary on what is known on the topic.

A whole blood test to measure SARS-CoV-2-specific response in COVID-19 patients.

OBJECTIVES: To examine whether specific T-cell-responses to SARS-CoV-2 peptides can be detected in COVID-19 using a whole-blood experimental setting, which may be further explored as a potential diagnostic tool. METHODS: We evaluated interferon (IFN)-γ levels after stimulating whole-blood with spike and remainder-antigens peptides megapools (MP) derived from SARS-CoV-2 sequences; interleukin (IL)-1ß, IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12p70, IL-13, IL-15, IL-17A, eotaxin, basic fibroblast growth factor (FGF), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), IFN-γ, Interferon gamma-induced protein 10 (IP-10), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1α, MIP-1ß, Platelet-derived growth factor (PDGF), RANTES (regulated on activation, normal T cell expressed and secreted), tumour necrosis factor-alpha (TNF-α), vascular endothelial growth factor (VEGF) were also evaluated. RESULTS: IFN-γ-response to spike and remainder-antigens MPs was significantly increased in 35 COVID-19 patients compared with 29 'no COVID-19' individuals (medians spike-MP: 0.26 vs 0, p = 0.0002; medians remainder-antigens-MP: 0.07 vs 0.02; p = 0.02). This response was detected independently of patients' clinical parameters. IFN-γ-response to SARS-CoV-2-unrelated antigens cytomegalovirus (CMV) and Staphylococcal Enterotoxin B (SEB) was similar in COVID-19 compared with 'no COVID-19' individuals (median CMV: 3.46 vs 5.28, p = 0.16; median SEB: 12.68 vs 15.05; p = 0.1). In response to spike-MPs in COVID-19- compared with 'no COVID-19' -individuals, we found significant higher median of IL-2 (50.08 vs 0, p = 0.0018), IFN-γ (90.16 vs 0, p = 0.01), IL-4 (0.52 vs 0, p = 0.03), IL-13 (0.84 vs 0, p = 0.007) and MCP-1 (4602 vs 359.2, p = 0.05). CONCLUSIONS: Immune response to SARS-CoV-2 peptides in a whole-blood assay is associated with COVID-19 and it is characterized by both Th1 and Th2 profile. This experimental approach may be useful for developing new T-cell based diagnostic tests for disease and vaccine settings.