PURPOSES

1. Determine the frequency of circulating immune cells (i.e. T cells, B cells, Neutrophils,
Monocytes absolute numbers) in COVID-19 patients at Hospital Trust of Verona.

2. Determine the plasma levels of soluble factors (i.e. IL-1beta, IL-6, IL-10, TNFalfa) in
COVID-19 patients at Hospital Trust of Verona.

3. Determinate the immune composition of lung and blood of COVID-19 patients that will be
enrolled at Hospital Trust of Verona through single-cell sequencing analysis, capable of
detecting also the viral sequences in each leukocyte populations.

4. Determine any potential links between cytokine storm, immune cells composition and
clinical parameters (i.e.) in COVID-19 patients

5. Profile patients with a different stage of disease to identify potential biomarkers

6. Identify SARS-CoV-2-associated sequences in immune cells

7. To define differences in immune cell composition the immune profiling will be done
before and after patients treatment used as part of clinical care in COVID-19 patients.

HYPOTHESES

1. Primary: Based on previous reports, COVID-19 patients showed significant immunological
alterations that need to be deeply investigated. Therefore, the main objective of this
study is to perform a phenotypic characterization of patients with COVID-19

2. Secondary: Immunological disorder induced by the infection can generate a systemic
pathology. Therefore, a) the investigators will identify potential immune-associated
biomarkers for rapid testing; b) the investigators will identify specific viral
sequences inside immune cells that can explain the systemic disease c) the investigators
will define immunological parameters that correlate with disease progression

JUSTIFICATION

The pandemic spread of a novel, highly pathogenic coronavirus (SARS-CoV-2) has found the
international medical community largely unprepared on prophylactic and therapeutic measures.
The resulting syndrome, known as COVID-19, is characterized by a profound dysfunction of the
upper and lower respiratory tract, with severity ranging from mild to moderate respiratory
failure, up to acute respiratory distress syndrome (ARDS) that is generally fatal2. Recently,
the crucial role of the "cytokine release syndrome (CRS), also referred to as "cytokine
storm", in acute lung damage and ARDS4,5 has become evident, thus providing the theoretical
ground for therapeutic approaches able to interfere with the inflammatory cascade. Indeed,
while the majority of patients either asymptomatic or with early stage of the disease are
able to clear the infection, some patients with moderate disease, requiring hospital
admittance, progress to a clinically severe phase associated with the "cytokine storm" within
10 days from symptom onset.

Delineation of a link between clinical parameters and immunological profile would provide a
rational to define the perfect treatment to reduce the incidence of COVID-19 associated ARDS
and mortality.

OBJECTIVES

Better understand 1) the link between immunological modifications and ARDS in COVID-19
patients; 2) the biomarker profile of COVID-19 patients with mild and severe disease; 3) the
impact of virus sequences in circulating immune cells on clinical outcomes in COVID-19
patients

RESEARCH DESIGN

Patients admitted to Hospital Trust of Verona will be isolated in Infective Unit or intensive
care unit (ICU) on basis of the . Here, specimens will be collected for laboratory
confirmation of SARS-CoV-2 using nasopharyngeal swab assessed PCR by the Microbiology and
Virology Unit. Clinical care will commence following the discretion of attending physicians
in accordance with Hospital Trust of Verona current operating procedures for the management
of COVID-19.

Once clinical care has commenced, a blood sample will be collected in three different
moments: T0 = at diagnosis (which is equivalent for severe symptoms that will begin a
therapeutic course, or for light symptoms within 72 hours from the COVID-19 diagnosis) ; T 1=
after 7 days from diagnosis (for patients admitted to the UOC the collection will be carried
out at the Operative Unit while for asymptomatic patients this collection will be done at the
patient's home by health workers and then the sample will be sent to the Immunology Section);
T2 = after 14 days from diagnosis similar to that described for the previous collection. All
the samples will be collected into vacutainer serum separator tubes (SST, Vacutainer®, Becton
& Dickinson). In some cases, patients admitted to ICU also receive BAL (bronchiole-alveolar
lavage) or tracheal / mini BAL aspiration. The BAL will be collected whenever it is an
integral part of the patient's diagnostic path or within thirty minutes of the patient's
confirmed death. In all cases, together with the BAL it will also be necessary to collect a
test tube of blood, independently and in addition to the samples described above.

These samples will be analyzed for: a) a complete immune phenotype by flow cytometry; b) a
panel of soluble factots (i.e. cytokines), c) a deep immunological composition by single-cell
RNA sequencing, d) presence of viral sequences.

Analysis will be performed at Immunology Section (a,b,c) and Microbiology and Virology
Section of Hospital Trust of Verona.

Immunophenotype analysis on whole peripheral blood will be performed according to a standard
no-wash procedure, using a PrepPlus™ 2 workstation (Beckman Coulter Inc., Brea, CA, USA).
Blood samples will be incubated with antibodies (Beckman Coulter Inc., Brea, CA, USA) for 15
min at 20°C. Following is the list of antibodies used:

- FITC-conjugated CD57 (clone NC1), PE-conjugated CD45RA (ALB11), ECD-conjugated CD8
(SFCI21Thy2D3), PC5.5-conjugated CD56 (N901), PC7-conjugated CD4 (SFCI12T4D11),
APC-conjugated CD27 (1A4CD27), APC A700-conjugated CD45 (J33), APC A750-conjugated CD3
(UCHT1), PB-conjugated CD16 (3G8), KR OR-conjugated CD19 (J3-119)

- FITC-conjugated CD57 (clone NC1), PE-conjugated HLA-DR (Immu-357), ECD-conjugated CD8
(SFCI21Thy2D3), PC5.5-conjugated CD38 (LS198-4-3), PC7-conjugated CD4 (clone
SFCI12T4D11), APC-conjugated CD14 (RMO52), APC A700-conjugated CD45 (J33), APC
A750-conjugated CD3 (UCHT1), PB-conjugated CD16 (3G8), KR OR-conjugated CD19 (J3-119)

- PE-conjugated IgD (IA6-2), ECD-conjugated CD3 (UCHT1), PC5.5-conjugated CD27 (1A4CD27),
PC7-conjugated CD20 (clone B9E9), APC-conjugated IgM (SA-DA4), APC A700-conjugated CD45
(J33), APC A750-conjugated CD38 (LS198-4-3), PB-conjugated CD21 (BL13), KR OR-conjugated
CD19 (J3-119) Red blood cell lysis and cell fixation will be performed using TQ-Prep
workstation and ImmunoPrep reagent system (Beckman Coulter Inc., Brea, CA, USA). Samples
will be acquired with Navios and analyzed with Navios software (Beckman Coulter Inc.,
Brea, CA, USA).

For detection of cytokines, sera of COVID-19 patients were tested for GM-CSF, G-CSF, M-CSF,
IFN-γ, IFN-α, IL-1, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17A,
IL-17F, IL-17E, IL-21, IL-22, IL-23, IL-27, IL-28A, IL-31, IL-33, IL-34, MIP-3α/CCL20, CCL2,
TNF-α, TNF-β, TGFβ production by Human ProcartaPlex™ Panel 1 multiplex (ThermoFisher
Scientific, Waltham, MA, USA).

BAL cells and circulating immune cells will be purified and cell counts will be adjusted
following 10xgenomics protocol. Briefly, the cell suspension was super-loaded with 50,000
cells, in the ChromiumTM Controller for partitioning single cells into nanoliter-scale Gel
Bead-In-Emulsions (GEMs). Single Cell 3' reagent kit v3.1 will be used for reverse
transcription, cDNA amplification and library construction of the gene expression libraries
(10x Genomics) following the detailed protocol provided by 10xGenomics. Hashtag libraries
will be prepared according to the cell hashing protocol for 10x Single Cell 3' Reagent Kit
v3.1. Biometra Trio Thermal Cycler will be used for amplification and incubation steps
(Analytik Jena). Libraries will be quantified by QubitTM 2.0 Fluorometer (ThermoFisher) and
quality will be checked using 2100 Bioanalyzer with High Sensitivity DNA kit (Agilent).
Sequencing will be performed in paired-end mode with a S1 and S2 flow cell (2 × 50 cycles
kit) using NovaSeq 6000 sequencer (Illumina).

The short history of this disease outbreak makes robust statistical power testing difficult.
However, for analysis during the treatment the investigators will consider only those
patients who are still alive, for whom the clinical and laboratory data for at least two time
points are available. Analysis on the duration of hospitalization will be performed in all
the patients enrolled except for the ones who died. Quantitative variables will be expressed
as the median and interquartile range (IQR), qualitative ones as percentages. To perform
pairwise comparisons, significance of difference will be evaluated by the Mann-Witney U-test
for quantitative variables and Fisher's exact test for categorical variables. The Wilcoxon
matched-pairs signed-rank test will be used to test the equality of matched pairs of
observation. Multiple logistic-regression analysis will be performed to assess the
association between the treatment and mortality for known negative prognostic factor (age,
sex and D-dimer). The strength of the association will be expressed by the Hazard Ratio (HR)
with 95% confidence interval (CI). Statistical significance will set at p-value <0.05, and
clinical analyses will be performed using statistical software STATA version 16.0 (StataCorp,
College Station, TX, USA). For statistical laboratory analyses the investigators will perform
paired comparisons by Student's t-test and one-way Anova test for repeated measures, using
Graph Pad Prism (San Diego, California, version 8.4.2).

All biological materials will be stored after anonymisation at of Medicine Department of
University and Hospital Trust of Verona for the duration of the study (8 months). After that,
all biological materials will be transferred at Biobanca of University and Hospital Trust of
Verona. All patients data will be only accessed by researchers involved in the study with a
personnel account. All patients are anonymzed during immunological analysis. All data are
protected by software and database of University and Hospital Trust of Verona.

The ownership of the data will only be referred to the Principal Investigator (PI) of the
project.

TIMELINE OF THE PROJECT Duration of specimen collection: 6 months. This period was estimated
on the need to treat patients with COVID-19 immediately Expected duration for the analysis of
the data obtained: 2 months. This period has been estimated from the investigators'
experience and includes both the analysis of the samples and the statistical analysis of the
data obtained.

Total duration of the project: 8 months