Immune Cells Phenotypes During COVID-19

Study Overview
=================
Brief Summary
-----------------
The ongoing pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS CoV-2) has infected more than one hundred twenty million peoples worldwide one year after its onset with a case-fatality rate of almost 2%. The disease due to the coronavirus 2019 (i.e., COVID-19) is associated with a wide range of clinical symptoms. As the primary site of viral invasion is the upper respiratory airways, lung infection is the most common complication. Most infected patients are asymptomatic or experience mild or moderate form of the disease (80 %). A lower proportion (15%) develop severe pneumonia with variable level of hypoxia that may required hospitalization for oxygen therapy. In the most severe cases (5%), patients evolve towards critical illness with organ failure such as the acute respiratory distress syndrome (ARDS). At this stage, invasive mechanical ventilation is required in almost 70 % and the hospital mortality rises to 37 %. Immune cells are key players during SARS CoV-2 infection and several alterations have been reported including lymphocytes (T, B and NK) and monocytes depletion, and cells exhaustion. Such alterations were much more pronounced in patients with the most severe form of the disease. Beside, a dysregulated proinflammatory response has also been pointed out as a potential mechanism of lung damage. Finally, COVID-19 is associated with an unexpectedly high incidence of thrombosis which probably results from the viral invasion of endothelial cells. The investigators aim to explore prospectively the alterations of innate and adaptive immune cells during both the acute and the recovery phase of SARS CoV-2 pneumonia. Flow and Spectral cytometry will be used to perform deep subset profiling focusing on T, B, NK, NKT, gamma-gelta T, monocytes and dendritic cells. Each specific cell type will be further characterized using markers of activation/inhibition, maturation/differenciation and senescence as well as chemokines receptors. T-cell memory specificity will be explore using specific SARS CoV-2 pentamer. Platelet activation and circulating microparticles will be explore using flow cytometry. Serum SARS CoV-2 antibodies (IgA, IgM, IgG), serum cytokines, and serum biomarkers of alveolar epithelial and endothelial cells will be analyze using ELISA and correlate with the severity of the disease.

Official Title
-----------------
Alterations of Innate and Adaptive Immune Cells During the Course of SARS CoV-2 Pneumonia

Conditions
-----------------
Sars-CoV2, Innate Immunity, Immunization; Infection, Alveolar Lung Disease, Endothelial Dysfunction

Intervention / Treatment
-----------------
* Biological: Peripheral blood samples


Participation Criteria
=================
Eligibility Criteria
-----------------
Inclusion Criteria: Age > 18 y Laboratory confirmed SARS CoV-2 infection (positive RT-PCR). Ground-glass opacity on chest computed-tomography Time from hospital admission to inclusion < or equal to 72 h Exclusion Criteria: Pregnant Under legal restriction

Ages Eligible for Study
-----------------
Minimum Age: 18 Years

Sexes Eligible for Study
-----------------
All

Accepts Healthy Volunteers
-----------------
Accepts Healthy Volunteers

Study Plan
=================
How is the study designed?
-----------------

Arms and Interventions

| Intervention/Treatment |
| --- |
|Biological: Peripheral blood samples|Peripheral blood samples at Day 0, Day 7, Day 14, Day 28, Day 90 and Day 180.|

What is the study measuring?
-----------------
Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Profiling of innate and adaptive immune cells during SARS CoV-2 infection. | Determination of cells population using spectral cytometry of PBMCs. | Day 0 | 
| Profiling of innate and adaptive immune cells during SARS CoV-2 infection. | Determination of cells population using spectral cytometry of PBMCs. | Day 7 | 
| Profiling of innate and adaptive immune cells during SARS CoV-2 infection. | Determination of cells population using spectral cytometry of PBMCs. | Day 14 | 
| Profiling of innate and adaptive immune cells during SARS CoV-2 infection. | Determination of cells population using spectral cytometry of PBMCs. | Day 28 | 
| Profiling of innate and adaptive immune cells during SARS CoV-2 infection. | Determination of cells population using spectral cytometry of PBMCs. | Day 90 | 
| Profiling of innate and adaptive immune cells during SARS CoV-2 infection. | Determination of cells population using spectral cytometry of PBMCs. | Day 180 | 
| Functional state of innate and adaptive immune cells during SARS CoV-2 infection. | Determination of the functional state of immune cells using spectral cytometry | Day 0 | 
| Functional state of innate and adaptive immune cells during SARS CoV-2 infection. | Determination of the functional state of immune cells using spectral cytometry | Day 7 | 
| Functional state of innate and adaptive immune cells during SARS CoV-2 infection. | Determination of the functional state of immune cells using spectral cytometry | Day 14 | 
| Functional state of innate and adaptive immune cells during SARS CoV-2 infection. | Determination of the functional state of immune cells using spectral cytometry | Day 28 | 
| Functional state of innate and adaptive immune cells during SARS CoV-2 infection. | Determination of the functional state of immune cells using spectral cytometry | Day 90 | 
| Functional state of innate and adaptive immune cells during SARS CoV-2 infection. | Determination of the functional state of immune cells using spectral cytometry | Day 180 | 
| Serum IgA, IgM and IgG antibodies during SARS CoV-2 infection. | Measurement of serum SARS CoV-2 IgA, IgM and IgG antibodies using Elisa. | Day 0 | 
| Serum IgA, IgM and IgG antibodies during SARS CoV-2 infection. | Measurement of serum SARS CoV-2 IgA, IgM and IgG antibodies using Elisa. | Day 7 | 
| Serum IgA, IgM and IgG antibodies during SARS CoV-2 infection. | Measurement of serum SARS CoV-2 IgA, IgM and IgG antibodies using Elisa. | Day 14 | 
| Serum IgA, IgM and IgG antibodies during SARS CoV-2 infection. | Measurement of serum SARS CoV-2 IgA, IgM and IgG antibodies using Elisa. | Day 28 | 
| Serum IgA, IgM and IgG antibodies during SARS CoV-2 infection. | Measurement of serum SARS CoV-2 IgA, IgM and IgG antibodies using Elisa. | Day 90 | 
| Serum IgA, IgM and IgG antibodies during SARS CoV-2 infection. | Measurement of serum SARS CoV-2 IgA, IgM and IgG antibodies using Elisa. | Day 180 | 
| Platelet activation and circulating microparticles assessment during SARS CoV-2 infection. | Determination of platelet activation and circulating microparticles levels using flow cytometry. | Day 0 | 
| Platelet activation and circulating microparticles assessment during SARS CoV-2 infection. | Determination of platelet activation and circulating microparticles levels using flow cytometry. | Day 7 | 
| Platelet activation and circulating microparticles assessment during SARS CoV-2 infection. | Determination of platelet activation and circulating microparticles levels using flow cytometry. | Day 14 | 
| Platelet activation and circulating microparticles assessment during SARS CoV-2 infection. | Determination of platelet activation and circulating microparticles levels using flow cytometry. | Day 28 | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Serum concentration of Pro-inflammatory and Anti-inflammatory cytokines in response to SARS CoV-2 infection. | Measurement of IL1β, IL-6, IL-10, IL-17A, IL-18, TNFα, IFNγ, CRTP-6 using Elisa. | Day 0 | 
| Serum concentration of Pro-inflammatory and Anti-inflammatory cytokines in response to SARS CoV-2 infection. | Measurement of IL1β, IL-6, IL-10, IL-17A, IL-18, TNFα, IFNγ, CRTP-6 using Elisa. | Day 7 | 
| Serum concentration of Pro-inflammatory and Anti-inflammatory cytokines in response to SARS CoV-2 infection. | Measurement of IL1β, IL-6, IL-10, IL-17A, IL-18, TNFα, IFNγ, CRTP-6 using Elisa. | Day 14 | 
| Serum concentration of Pro-inflammatory and Anti-inflammatory cytokines in response to SARS CoV-2 infection. | Measurement of IL1β, IL-6, IL-10, IL-17A, IL-18, TNFα, IFNγ, CRTP-6 using Elisa. | Day 28 | 
| Serum concentration of Pro-inflammatory and Anti-inflammatory cytokines in response to SARS CoV-2 infection. | Measurement of IL1β, IL-6, IL-10, IL-17A, IL-18, TNFα, IFNγ, CRTP-6 using Elisa. | Day 90 | 
| Serum concentration of Pro-inflammatory and Anti-inflammatory cytokines in response to SARS CoV-2 infection. | Measurement of IL1β, IL-6, IL-10, IL-17A, IL-18, TNFα, IFNγ, CRTP-6 using Elisa. | Day 180 | 
| Serum alveolar epithelial and endothelial cells biomarkers during SARS CoV-2 infection. | Measurement of KL-6, CC-16, S-RAGE, ANG-2 using ELISA. | Day 0 | 
| Serum alveolar epithelial and endothelial cells biomarkers during SARS CoV-2 infection. | Measurement of KL-6, CC-16, S-RAGE, ANG-2 using ELISA. | Day 7 | 
| Serum alveolar epithelial and endothelial cells biomarkers during SARS CoV-2 infection. | Measurement of KL-6, CC-16, S-RAGE, ANG-2 using ELISA. | Day 14 | 
| Serum alveolar epithelial and endothelial cells biomarkers during SARS CoV-2 infection. | Measurement of KL-6, CC-16, S-RAGE, ANG-2 using ELISA. | Day 28 | 
| Kinetic of surface biomarkers expression on neutrophils (C64) and monocytes (CD169, HLA-DR) during SARS CoV-2 infection. | Measurement of nCD64, mCD169 and mHLA-DR using the VersaPOC one-step rapid flow cytometry method. | Day 0 | 
| Kinetic of surface biomarkers expression on neutrophils (C64) and monocytes (CD169, HLA-DR) during SARS CoV-2 infection. | Measurement of nCD64, mCD169 and mHLA-DR using the VersaPOC one-step rapid flow cytometry method. | Day 1 | 
| Kinetic of surface biomarkers expression on neutrophils (C64) and monocytes (CD169, HLA-DR) during SARS CoV-2 infection. | Measurement of nCD64, mCD169 and mHLA-DR using the VersaPOC one-step rapid flow cytometry method. | Day 2 | 
| Kinetic of surface biomarkers expression on neutrophils (C64) and monocytes (CD169, HLA-DR) during SARS CoV-2 infection. | Measurement of nCD64, mCD169 and mHLA-DR using the VersaPOC one-step rapid flow cytometry method. | Day 3 | 
| Kinetic of surface biomarkers expression on neutrophils (C64) and monocytes (CD169, HLA-DR) during SARS CoV-2 infection. | Measurement of nCD64, mCD169 and mHLA-DR using the VersaPOC one-step rapid flow cytometry method. | Day 5 | 
| Kinetic of surface biomarkers expression on neutrophils (C64) and monocytes (CD169, HLA-DR) during SARS CoV-2 infection. | Measurement of nCD64, mCD169 and mHLA-DR using the VersaPOC one-step rapid flow cytometry method. | Day 7 | 
| Kinetic of surface biomarkers expression on neutrophils (C64) and monocytes (CD169, HLA-DR) during SARS CoV-2 infection. | Measurement of nCD64, mCD169 and mHLA-DR using the VersaPOC one-step rapid flow cytometry method. | Day 9 | 
| Kinetic of surface biomarkers expression on neutrophils (C64) and monocytes (CD169, HLA-DR) during SARS CoV-2 infection. | Measurement of nCD64, mCD169 and mHLA-DR using the VersaPOC one-step rapid flow cytometry method. | Day 11 | 
| Kinetic of surface biomarkers expression on neutrophils (C64) and monocytes (CD169, HLA-DR) during SARS CoV-2 infection. | Measurement of nCD64, mCD169 and mHLA-DR using the VersaPOC one-step rapid flow cytometry method. | Day 14 | 
| Kinetic of surface biomarkers expression on neutrophils (C64) and monocytes (CD169, HLA-DR) during SARS CoV-2 infection. | Measurement of nCD64, mCD169 and mHLA-DR using the VersaPOC one-step rapid flow cytometry method. | Day 17 | 
| Kinetic of surface biomarkers expression on neutrophils (C64) and monocytes (CD169, HLA-DR) during SARS CoV-2 infection. | Measurement of nCD64, mCD169 and mHLA-DR using the VersaPOC one-step rapid flow cytometry method. | Day 21 | 
| Kinetic of surface biomarkers expression on neutrophils (C64) and monocytes (CD169, HLA-DR) during SARS CoV-2 infection. | Measurement of nCD64, mCD169 and mHLA-DR using the VersaPOC one-step rapid flow cytometry method. | Day 28 |