COVID-19 Clinical Trial
Official title:
Study of Immune-mediated Mechanisms in Patients Tested Positive for SARS-CoV-2: Phenotypic and Functional Analysis of Monocytes and NK Cells in the Blood of Subjects Affected by Covid 19
SARS-CoV-2 belong to beta-coronavirus family and its transmission route and symptoms follow
those of all community-acquired coronaviruses. The main difference of the novel Coronavirus
is the higher mortality rate, that is around 3%.
Death rate is over 1% only for patients over 50 years old, whereas until 40 years old is
under 0,4%. No fatalities are declared among children under 10 years old to date. Death rate
is almost double for male rather than female. This distribution of mortality rate according
to age of infected patients could be only partially ascribed to other comorbidities in
addition to great age. In fact, patients with no pre-existing conditions have however a case
fatality rate of 0,9%.
The almost null rate of severe illness in children and generally in patients younger than 40
years old is quite un-explicable. Infant, children and young people could be infected but
infection is rapidly self-limited or without symptoms. Older patients undergo severe lung
injury as consequence of an immune response that is late in coming.
Possible explanation of these phenomena could be something, which assure ability to prompt
response to SARS-CoV-2 in younger people independently from the novelty of the virus itself.
It would seem to be that younger people are already sensitized to the antigens of the virus
without a previous contact.
This immunity is not really specific, but "partially specific" for many antigens of the
virus, however able to limit the infection in the organism. Something stimulated the immune
system and it scattered immunity against more and more antigens present. Children are the age
group mostly exposed to all community-circulating viruses.
This immunity is not persistent but progressively fade out. It protects from the age of two,
when the hypothetical stimulation occurs, to the fifth decade because of its slow decrease.
The only external stimulation, which healthy people receive are vaccines. All vaccinations
and especially tetanic, diphtheria toxoids and inactivated bacteria as pertussis could
stimulate immune system. They develop the specific immunity but generate also a sprouting
immunity against antigens in transit, as coronaviruses and other community-circulating
viruses.
The developed immunity gives some protection against multiple viral infection for years until
the natural fade out.
After the fifth decade, that immunity is slower to be recall and reactivated. Additionally,
transplant recipients and HIV infected patients, which have an immune system inhibited,
unexpectedly, do not seem to suffer the worst complications of SARS-CoV-2 infection. An
immune system imbalance could be play a pivotal role during the reaction to the virus,
limiting destructive consequences of excessive inflammation.
According to the medical hypothesis on which the protocol is based on, young people could
benefit from a functional adaptation of innate immune cells induced through epigenetic
reprogramming and, especially, a pre-existing "partially specific" immunity to the community
viruses caused by "bystander effect" of preceding vaccinations. In this study, we will
explore the main differences existing among patients infected by SARS-CoV-2 who experience
the illness at different degree of severity. We suppose to recognize different populations of
patients, each one with a specific immunological pattern. It could differ in terms of
cytokines, soluble factors serum level and immune cells activity both of the innate
compartment and of the acquired one. The proof of a role of these immunological phenomena in
the pathogenesis of Covid-19 are bases for implementation of therapeutic immunomodulatory
treatments. In addition, the definition of an immunological risk profile could tailor
established therapies to each kind of patient.
According to the medical hypothesis on which the protocol is based on, young people could
benefit from a functional adaptation of innate immune cells induced through epigenetic
reprogramming and, especially, a pre-existing "partially specific" immunity to the community
viruses caused by "bystander effect" of preceding vaccinations. In this study, we will
explore the main differences existing among patients infected by SARS-CoV-2 who experience
the illness at different degree of severity. We suppose to recognize different populations of
patients, each one with a specific immunological pattern. It could differ in terms of
cytokines, soluble factors serum level and immune cells activity both of the innate
compartment and of the acquired one.
Data will be collected using 3 approaches:
- An experimental analysis, for clusters of patients, to study the innate immune response
and to identify the genetic profiles.
- An epidemiological analysis, in order to identify the patients' vaccination history;
- A clinical analysis, to detect the immunological profile;
For the specific analysis, to study the innate immune response and to identify the genetic
profiles, scientists will analyze, from the peripheral blood, NK cells, monocytes, CD4 and
CD8 T cells of both groups: healthy patients (tested negative for SARS-CoV-2) and sick
patients of the subgroups (AS 19, PAU19, POL19, ARD19).
From different groups of patients, blood samples (10-15 mL) will be drawn into EDTA tubes,
centrifuged at 360 g for 10 minutes to obtain plasma that it will be stored at -80°C for
subsequent analysis for cytokines and chemokines of interest by ELISA (IL-1b, IL-6, TNF,
IFN-a, IL-10, IL-12, CCL2 and CXCL10) at the end of enrolment.
The cell pellets will be brought back to the initial volume with PBS and diluted 1:1 (v/v),
and then subjected to a density gradient stratification with Ficoll Histopaque-1077, at 500 g
for 30 minutes. The Peripheral Blood Mononuclear Cells (PBMCs) derived from the white ring
will be collected, washed twice in PBS, and then used for subsequent experiments using a flow
cytometer assay (REFF). The in vitro culture using PBMCs can vary from ex vivo 1 day to a few
days, and cells will be maintained in RPMI 1640 medium, supplemented with 10% fetal bovine
serum (FBS), 2 mM l-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin (P/S), at 37°C,
5% CO2.
To perform ex vivo fluorescence-activated cell sorter (FACS) phenotype analysis, 2.5x105 of
fresh total PBMCs per FACS tube will be stained for 30 minutes at 4°C with monoclonal
antibodies (mAbs) as follows: CD3-PerCP, CD56-APC, CD16-FITC, NKG2A-PE, NKG2C-PE, NKGD2-PE,
DNAM-1-PE, CD25-PE, CD69-PE. Following Forward/Side Scatter setting, NK cells will be
identified into two cell subsets, i.e. as CD3- and CD56dim CD16+ cells (CD56dim NK cells, the
major subset, about 90%), and CD3- and CD56bright CD16-/low cells (CD56bright NK cells, the
minor subset, about 10%). Other markers expression will be evaluated on both subsets of gated
cells.
For ex vivo FACS evaluation of monocytes phenotype analysis, 2.5x105 of fresh total PBMCs per
FACS tube will be stained for 30 minutes at 4°C with mAbs as follows: CD45-APC, CD14-FITC and
CD16-V450, PE-CD209, PE-CD80. Following Forward/Side Scatter setting, monocytes will be
identified into three subsets, i.e. as CD14+ and CD16- cells (the main subset, about 90%),
CD14+ CD16+ (the minor subset, about 10%), and CD14-/low CD16+ (the other minor subset).
We will also evaluate phenotype for CD4 and CD8 T cells and CD4/CD8 T ratio using 2x105 of
fresh total PBMCs, as previously described, with following mAbs: CD3-PerCP, CD4-APC, CD8-V450
as well as T regulatory (Treg) cells, as CD3-PerCP, CD4-APC, CD25-PE.
PBMCs will be also in vitro stimulated for 4 h with different types of stimuli, such as LPS
(recognized by the TLR4), poly(I:C) (recognized by the TLR3), poly (I:C) plus IL-2 plus
IL-12, and phorbol myristate acetate (PMA) plus ionomycin in presence of monensin and
brefeldin. This procedure will allow studying specific cytokine/chemokine production from NK
cells and monocytes by using a FACS intracellular assay, as described previously (REFF). For
NK cells, we will measure IFN-a-PE, TNF-PE, CCL2-PE, CXCL10-PE and CD107a-PE (degranulation
marker). For monocytes we will investigate: IL-6-PE, TNFa-PE, IL-12-PE, CXCL10-PE (M1-type
pro-inflammatory markers) and TGFb-PE, IL-10-PE, CCL18-PE (M2-type anti-inflammatory markers)
Whenever the quantity of PBMCs is sufficient, other functional in vitro tests on NK cells and
monocytes will be set up. In particular, PBMCs will be studied for 4 h NK cell
degranulation/cytotoxic function towards erytroleucemic K562 cell line assessing surface
CD107a-PE using a flow cytofluorimetric assay (REFF).
Whenever the quantity of PBMCs is sufficient, monocytes will be purified using anti-CD14
microbeads with magnetic separator and NK cells with RosetteSep kit to obtain >90% purified
cell populations.
Purified monocytes and purified NK could be maintained separated or together in culture using
RPMI 1640 medium with 10%FBS, and supplemented with M-CSF and IL-2, respectively, stimulated
with different stimuli (see above), and then checked for intracellular cytokines/chemokines
of interest (see above). At the same time the supernatants (conditioned medium, CM) could be
harvested at the end of in vitro incubation culture and assessed for cytokines/chemokines
using ELISA (IL-1b, IL-6, TNFa, IFNa, IL-10, IL-12, CXCL10).
The 4-5 days in vitro culture of monocytes will be further stimulated 24 h with LPS plus IFNg
(M1 stimulus) or (IL-4) (M2 stimulus) to investigate macrophage polarization studying surface
M1 markers (TNF, CXCL10) or M2 markers (IL-10, CCL18) to check the prevalence of macrophage
polarization in different groups of Covid-19 patients.
The epidemiological analysis will be carried out integrating both vaccination history and the
data daily collected after hospital admission. ATS Insubria archives will provides missing
data.
Considering the immunological profile, patients with Covid-19 will be tested for routine
examinations and the following:
- lymphocyte immunophenotyping;
- determination of C3 and C4 complement fractions activity;
- immunoglobulin (IgG, IgM, IgA, IgE) serum level;
- serum protein electrophoresis;
- Angiotensin Converting Enzyme (ACE) serum level;
- CMV (Citomegalovirus) serology test;
- IL-6 serum level.
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