Clinical, Functional, Immunological and Genetic Factors on the Severity of the Course of Coronavirus Infection

Objectives of the program:

1. To determine the clinical and functional characteristics of patients with varying
degrees of the course of the acute phase of COVID-19 and Post Covid syndrome.

1.1. To study the features of neurological disorders in patients with varying degrees of
the course of the acute phase of COVID-19 and Post Covid syndrome.

2. To study the immunological profile of patients with varying degrees of the course of the
acute phase of COVID-19 and Post Covid syndrome.

3. To study the genetic profile of patients with varying degrees of the course of the acute
phase of COVID-19 and Post Covid syndrome.

4. Identify potential predictors of COVID-19 severity.

5. To determine the markers that allows predicting the development of the Post Covid
syndrome.

6. Based on the selected markers, develop a COVID-19 outcome scale to determine the tactics
of patient management to prevent the development of Post Covid syndrome.

The study will include patients with a positive PCR test for COVID-19. Patients in the acute
phase of the course of the disease are monitored and treated in accordance with the
republican COVID-19 treatment protocol. After signing the informed consent, the patient will
be included in the study. The collection of the necessary materials for subsequent analyzes
(clinical-functional, genetic, immunological) will be carried out in accordance with this
protocol. Subsequently, patients are observed within one year from the moment of illness in
accordance with the study protocol and with the collection of all necessary materials.

Clinical and functional analysis:

Detection of RNA of the COVID-19 virus using PCR analysis. Conducting complex laboratory
studies in accordance with table 1.

General blood analysis Complete blood count on an analyzer with differentiation of 5 classes
of cells, the ratio of neutrophils to lymphocytes

Blood chemistry ALT, AST, total bilirubin, direct, LDH, CRP, alpha-amylase, creatinine, urea,
glucose, ferritin, glycosylated hemoglobin, vitamin 25 - OH vitamin D, vitamin B12

Coagulogram D-dimers, fibrinogen, INR, APTT

Other NT-pro BNP, Homocysteine, IL 6, Troponin, blood group determination

Linked immunosorbent assay Determination of IgG and IgM antibodies to SARS-CoV-2 coronavirus
(COVID-19) in blood serum, RBD

Functional diagnostics • ECG

- EchoCG + strain

- CT scan of the lungs

- Holter

- SMAD

- Kidney ultrasound

- Ultrasound

- Doppler ultrasonography of veins and arteries

- Chalder Scale, EQ Questionnaire Table 1. Clinical and functional analysis

Neurological disorders:

1. Neurological examination with the isolation of neurological syndromes (motor, cognitive
impairments, sleep disorders, asthenic-depressive syndromes, etc.).

2. Neuropsychological methods - research on the scales of anxiety and depression, MMSE,
etc.

3. Instrumental method - EEG, polysonography, ultrasound of the neck vessels, CT perfusion.

4. Laboratory research methods:

1. The study of antibodies to some neurospecific antigens - myelin basic protein
(MBP), neurospecific enolase (NSE).

2. Study of cellular immunity (CD3 +, CD4 +, CD8 +) and general indicators of humoral
immunity (IgG, IgA, IgM, circulating immune complexes).

Immunological analysis:

A comprehensive immunological analysis will be carried out to determine the level of the
immune response. Calculation of the level of CD4 +, CD8 + and NK cells. The level of
antibodies of the IgG and IgM classes to the proteins of the coronavirus S1, RBD and N was
determined Multiplex Immunoassay. For evaluation of immunological parameters, samples are
diluted in 200 μl of phosphate buffer, centrifuged and the supernatant analyzed using the
manufacturer's protocol. The MILLIPLEX MAP human cytokine / chemokine magnetic bead panel
will be used for the analysis of multiple cytokines and chemokines / immunoglobulins, and the
Milliplex® magnetic bead panel (HGAMMAG-301K-06, EMD Millipore Corp., Billerica, MA) will be
used for immunoglobulin isotyping. Samples will be analyzed on Bioplex BIO-RAD for the
following indicators: sCD40L, EGF, Eotaxin / CCL11, FGF-2, Flt-3 ligand, Fractalkine, G-CSF,
GM-CSF, GRO, IFN-α2, IFN-γ, IL -1α, IL-1β, IL-1ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,
IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17A, IP-10, MCP-1, MCP-3, MDC
(CCL22), MIP-1α, MIP-1β, PDGF- AA, PDGF-AB / BB, RANTES, TGF-α, TNF-α, Immunophenotyping of
T-cell and B-cell subpopulations using the flow cytometry method. Subpopulations of B- and
T-lymphocytes will be examined by staining peripheral blood mononuclear cells (PBMCs)
isolated from whole peripheral blood with monoclonal antibodies conjugated to fluorochromes.
PBMCs can be isolated from EDTA-treated whole blood using Ficoll density gradient
centrifugation or special erythrocyte lysis buffers. It is preferable to analyze isolated
PBMCs directly on the day of blood collection, which gives more reliable results. PBMC
viability will be assessed using LIVE / DEAD ™ fixed dead cell kits or 0.4% trypan blue and
propidium iodide solution. After lysis of erythrocytes and incubation with monoclonal
antibodies, the stained cells are resuspended in a staining medium and examined using a MoFlo
Astrios flow cytometer (Beckman Coulter, USA). The resulting data will be analyzed using
Summit (Beckman Coulter) and FloJo (Tree Star) software.

Forward and side scatter will be used to distinguish the lymphocyte population in addition to
the signal from specific fluorochromes. Distribution CD3-, CD5 +, CD19 + (total number of
B-lymphocytes), CD5-, CD19 +, CD27 + (memory B-cells), CD19 + CD27- (naive B-cells), CD19 +
CD27 + CD38 + IgD - (Class-Switched Memory B-Cells) CD19 + CD27 + CD38 + IgD + (Unswitched
Memory B-Cells) will be analyzed on the general lymphocyte population. The distribution of
markers CD3, CD4 and CD8 will be analyzed in the pool of T-lymphocytes. To analyze the
differentiation status of T cells, cells are additionally stained with anti-CCR7, anti-CD45RO
antibodies. Antibodies will be purchased from Invitrogen ™ unless otherwise noted.

Genetic analysis:

Isolation (extraction) of DNA will be performed from whole blood using commercial kits
according to the manufacturer's instructions. To analyze a large number of genetic markers,
it is planned to carry out genome-wide sequencing followed by analysis of genetic
polymorphisms of candidate genes encoding coronavirus receptors and immunological factors.

Sequencing will be performed using high-throughput next generation sequencing platforms
Illumina NovaSeq6000 (Illumina), method validation using traditional capillary sequencing -
ABI 3730XL ™ DNA Analyzer (Life Technologies), real-time PCR.

Bioinformatic data analysis.Bioinformatics sequencing data will be analyzed. The software
packages for bioinformatic analysis of sequencing data (GATK, bwa, bowtie, bowtie2, VarScan
etc) will be used. The sequencing data will be compared with the publicly available data from
the world's international databases of genomic research (https://www.covid19hg.org/, ExAC,
HGMD (Human Gene Mutation Database), ESP, GeneBank, NCBI, ESP6500, 1000Genomes, SNPDb130,
Ensembl, ClinVar, SNPedia, etc.). Differences in the type and frequency of genomic variation
among the surveyed groups will be determined.

To classify the detected genetic variants, in silico models will be used (SIFT_score / pred,
Polyphen2_HDIVscore / pred, Polyphen2_HVAR_score / pred, LRT_score / pred,
MutationTaster_score / pred, MutationAssessor_score / pred, FATHMM_score / pred, Radial /
MetaRVM_score pred). The classification of clinically significant genetic variants will be
carried out according to the international ACMG / AMG criteria.

Statistical analysis:

Statistical analysis will be carried out using version R 3.6.2. Quantitative data, including
clinical, biochemical, molecular genetic parameters, will be checked for normality using the
Shapiro-Wilks test and recognized as parametric in distribution. Comparison of mean
differences will be performed using one-way ANOVA, and subsequent pairwise comparison will be
performed using Tukey's special test. Within-group mean differences will be performed using
the paired sample t-test. The graphs will be executed using the ggplot2 R package.
Statistical analysis will be performed for the multiplex analysis results using the R psych
package and standard t-tests.

Inspection frequency:

Patients are followed up for 12 months from the date of illness. Disease detection
corresponds to the baseline (day 0). At the time of diagnosis, materials are taken for
clinical, functional and immunological diagnostics. After that, the sampling is carried out
every month for the next year from the moment of the disease in accordance with the study
protocol (Figure 2). The collection of materials for genetic analysis is carried out once.