Cellular-Mediated Immunity in COVID-19

Study Overview
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Brief Summary
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In order to prevent reinfection, it is needed to detect the cellular-mediated immune response to the Sars-CoV-2 infection. The first goal of this study will be to detect the cellular-mediated immune response in patients affected by COVID-19 (with or without vaccination) and healthy subjects who undergone vaccination program. The second goal of this study will be to identify the genetic and epigenetic biomarkers that influence individual immunological response and clinical evolution to the severe manifestations of the COVID-19.

Detailed Description
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First goal: Characterization of the immune response using detailed phenotypic evaluation of the adaptive compartment comprising B, T, and NK cells. The Investigators aim is to detect the cellular-mediated immune response in patients affected by COVID-19 and subjects who undergone vaccination program. The Investigators will recruit three classes of subjects: Patients with previous Sars-CoV-2 infection who did not undergo vaccination; Patients with previous Sars-CoV-2 infection who undergone vaccination; Subjects without previous Sars-CoV-2 infection who undergone vaccination. Second goal: Evaluation of genetic and epigenetic biomarkers that influence individual immunological response and susceptibility to thromboembolic manifestations in severe COVID-19 hospitalized patients. The Investigators will recruit: A total of 50 severe COVID-19 patients admitted to the Sub-Intensive Care Unit of A.O.R.N. Ospedali dei Colli, Cotugno Hospital, Naples (Italy) of which N=50 with thromboembolic complications (PE+) and N=50 without thromboembolic complications (PE-); A total of N=50 healthy subjects (never diagnosed with Sars-Cov2 infection) among the volunteer blood donors attending the U.O.C. Divisione di Immunologia Clinica, Immunoematologia, Medicina Trasfusionale e Immunologia dei Trapianti, Dipartimento di Medicina Interna e Specialistica, AOU, L. Vanvitelli University of Campania (Naples, Italy)

Official Title
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DEtection of Cellular-MEdiated immuniTy in COVID-19 Patients and Subjects Who undeRgone vAccination Program

Conditions
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Covid19, Thromboembolism

Intervention / Treatment
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* Diagnostic Test: Multitest 6 color TBNK and BD Lyotubes
* Biological: genomicDNA extraction


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Age > 18 years Previous diagnosis of COVID-19 Absence of COVID-19 diagnosis Exclusion Criteria: Age <18 years Inflammatory diseases

Ages Eligible for Study
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Minimum Age: 18 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Severe COVID-19 hospitalized patients<br>A total of 50 severe COVID-19 patients admitted to the Sub-Intensive Care Unit of A.O.R.N. Ospedali dei Colli, Cotugno Hospital, Naples (Italy) will be recruited, of which N=50 with thromboembolic complications (PE+) and N=50 without thromboembolic complications (PE-) balanced for age and sex of individuals. | Biological: genomicDNA extraction<br>* Peripheral blood biospecimens (about 10-15 mL) will be collected through venipuncture in EDTA tubes. Peripheral blood mononuclear cells (PBMCs) will be isolated using Ficoll® Paque Plus (Sigma-Aldrich) centrifugation gradient and genomic DNA will be extracted from fresh PBMCs using DNeasy Blood & Tissue kit (QIAGEN), according to manufacturer protocols<br>|
| Healthy controls (comparator group)<br>As control group (CTRL), we will recruit a total of N=50 healthy subjects (never diagnosed with Sars-Cov2 infection) among the volunteer blood donors attending the U.O.C. Divisione di Immunologia Clinica, Immunoematologia, Medicina Trasfusionale e Immunologia dei Trapianti, Dipartimento di Medicina Interna e Specialistica, AOU, L. Vanvitelli University of Campania (Naples, Italy). | Biological: genomicDNA extraction<br>* Peripheral blood biospecimens (about 10-15 mL) will be collected through venipuncture in EDTA tubes. Peripheral blood mononuclear cells (PBMCs) will be isolated using Ficoll® Paque Plus (Sigma-Aldrich) centrifugation gradient and genomic DNA will be extracted from fresh PBMCs using DNeasy Blood & Tissue kit (QIAGEN), according to manufacturer protocols<br>|
| COVID-19 patients compared with vaccinated subjects<br>Patients with previous Sars-CoV-2 infection who did not undergo vaccination; Patients with previous Sars-CoV-2 infection who undergone vaccination Subjects without previous Sars-CoV-2 infection who undergone vaccination | Diagnostic Test: Multitest 6 color TBNK and BD Lyotubes<br>* The Multitest 6 color TBNK allows us to count the population-specific B, T, NK cells<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Detection of Cellular-Mediated Immune Response | The Investigators will perform the BD Multitest 6-color TBNK by using 50 uL of peripheral blood to detect the absolute number (N) of CD3+, CD19+, CD16+, CD46+, CD4+ and CD8+ cells. | 3 Months | 
| Detection of Cellular-Mediated Immune Response | The Investigators will perform the BD™ Lyotubes kit by using 150 uL of peripheral blood to detect the absolute number (N) of CD4+ and CD8+ cells undergoing to differentiation from naive T cells to central memory, effector memory, and terminal memory | 3 Months | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Detection of T cell subpopulation maturation | The Investigators will perform the BD™ Lyotubes kit by using 150 uL of peripheral blood to detect the absolute number (N) of CD4+ and CD8+ cells undergoing to differentiation from naive T cells to central memory, effector memory, and terminal memory. | 5 months | 
| Detection of T cell subpopulation maturation | The Investigators will perform the BD™ Lyotubes kit by using 150 uL of peripheral blood to detect the % of CD4+ and CD8+ cells undergoing to differentiation from naive T cells to central memory, effector memory, and terminal memory. | 5 months |