<clinical_study>
  <!-- This xml conforms to an XML Schema at:
    https://clinicaltrials.gov/ct2/html/images/info/public.xsd -->
  <required_header>
    <download_date>ClinicalTrials.gov processed this data on September 20, 2023</download_date>
    <link_text>Link to the current ClinicalTrials.gov record.</link_text>
    <url>https://clinicaltrials.gov/ct2/show/NCT04438629</url>
  </required_header>
  <id_info>
    <org_study_id>IMMUNOVID 2590CESC</org_study_id>
    <nct_id>NCT04438629</nct_id>
  </id_info>
  <brief_title>Evaluation of Immune Response in COVID-19 Patients</brief_title>
  <acronym>IMMUNOVID</acronym>
  <official_title>Evaluation of Immune Response in COVID-19 Patients</official_title>
  <sponsors>
    <lead_sponsor>
      <agency>Azienda Ospedaliera Universitaria Integrata Verona</agency>
      <agency_class>Other</agency_class>
    </lead_sponsor>
  </sponsors>
  <source>Azienda Ospedaliera Universitaria Integrata Verona</source>
  <oversight_info>
    <is_fda_regulated_drug>No</is_fda_regulated_drug>
    <is_fda_regulated_device>No</is_fda_regulated_device>
  </oversight_info>
  <brief_summary>
    <textblock>
      The aim of the project is to evaluate the immunological features of COVID-19 patients.&#xD;
      Patients are recruited without any pharmacological treatments restriction. The number of&#xD;
      samples is estimated on the basis of feasibility, that means on the maximum number of&#xD;
      patients with COVID-19, who are expected to be able to be enrolled by the units involved.&#xD;
      Based on the investigators' experience, gained in the onco-immunological field, considering&#xD;
      the time and economic resources available, the investigators expect to enroll at least 80&#xD;
      patients.&#xD;
    </textblock>
  </brief_summary>
  <detailed_description>
    <textblock>
      PURPOSES&#xD;
&#xD;
        1. Determine the frequency of circulating immune cells (i.e. T cells, B cells, Neutrophils,&#xD;
           Monocytes absolute numbers) in COVID-19 patients at Hospital Trust of Verona.&#xD;
&#xD;
        2. Determine the plasma levels of soluble factors (i.e. IL-1beta, IL-6, IL-10, TNFalfa) in&#xD;
           COVID-19 patients at Hospital Trust of Verona.&#xD;
&#xD;
        3. Determinate the immune composition of lung and blood of COVID-19 patients that will be&#xD;
           enrolled at Hospital Trust of Verona through single-cell sequencing analysis, capable of&#xD;
           detecting also the viral sequences in each leukocyte populations.&#xD;
&#xD;
        4. Determine any potential links between cytokine storm, immune cells composition and&#xD;
           clinical parameters (i.e.) in COVID-19 patients&#xD;
&#xD;
        5. Profile patients with a different stage of disease to identify potential biomarkers&#xD;
&#xD;
        6. Identify SARS-CoV-2-associated sequences in immune cells&#xD;
&#xD;
        7. To define differences in immune cell composition the immune profiling will be done&#xD;
           before and after patients treatment used as part of clinical care in COVID-19 patients.&#xD;
&#xD;
      HYPOTHESES&#xD;
&#xD;
        1. Primary: Based on previous reports, COVID-19 patients showed significant immunological&#xD;
           alterations that need to be deeply investigated. Therefore, the main objective of this&#xD;
           study is to perform a phenotypic characterization of patients with COVID-19&#xD;
&#xD;
        2. Secondary: Immunological disorder induced by the infection can generate a systemic&#xD;
           pathology. Therefore, a) the investigators will identify potential immune-associated&#xD;
           biomarkers for rapid testing; b) the investigators will identify specific viral&#xD;
           sequences inside immune cells that can explain the systemic disease c) the investigators&#xD;
           will define immunological parameters that correlate with disease progression&#xD;
&#xD;
      JUSTIFICATION&#xD;
&#xD;
      The pandemic spread of a novel, highly pathogenic coronavirus (SARS-CoV-2) has found the&#xD;
      international medical community largely unprepared on prophylactic and therapeutic measures.&#xD;
      The resulting syndrome, known as COVID-19, is characterized by a profound dysfunction of the&#xD;
      upper and lower respiratory tract, with severity ranging from mild to moderate respiratory&#xD;
      failure, up to acute respiratory distress syndrome (ARDS) that is generally fatal2. Recently,&#xD;
      the crucial role of the &quot;cytokine release syndrome (CRS), also referred to as &quot;cytokine&#xD;
      storm&quot;, in acute lung damage and ARDS4,5 has become evident, thus providing the theoretical&#xD;
      ground for therapeutic approaches able to interfere with the inflammatory cascade. Indeed,&#xD;
      while the majority of patients either asymptomatic or with early stage of the disease are&#xD;
      able to clear the infection, some patients with moderate disease, requiring hospital&#xD;
      admittance, progress to a clinically severe phase associated with the &quot;cytokine storm&quot; within&#xD;
      10 days from symptom onset.&#xD;
&#xD;
      Delineation of a link between clinical parameters and immunological profile would provide a&#xD;
      rational to define the perfect treatment to reduce the incidence of COVID-19 associated ARDS&#xD;
      and mortality.&#xD;
&#xD;
      OBJECTIVES&#xD;
&#xD;
      Better understand 1) the link between immunological modifications and ARDS in COVID-19&#xD;
      patients; 2) the biomarker profile of COVID-19 patients with mild and severe disease; 3) the&#xD;
      impact of virus sequences in circulating immune cells on clinical outcomes in COVID-19&#xD;
      patients&#xD;
&#xD;
      RESEARCH DESIGN&#xD;
&#xD;
      Patients admitted to Hospital Trust of Verona will be isolated in Infective Unit or intensive&#xD;
      care unit (ICU) on basis of the . Here, specimens will be collected for laboratory&#xD;
      confirmation of SARS-CoV-2 using nasopharyngeal swab assessed PCR by the Microbiology and&#xD;
      Virology Unit. Clinical care will commence following the discretion of attending physicians&#xD;
      in accordance with Hospital Trust of Verona current operating procedures for the management&#xD;
      of COVID-19.&#xD;
&#xD;
      Once clinical care has commenced, a blood sample will be collected in three different&#xD;
      moments: T0 = at diagnosis (which is equivalent for severe symptoms that will begin a&#xD;
      therapeutic course, or for light symptoms within 72 hours from the COVID-19 diagnosis) ; T 1=&#xD;
      after 7 days from diagnosis (for patients admitted to the UOC the collection will be carried&#xD;
      out at the Operative Unit while for asymptomatic patients this collection will be done at the&#xD;
      patient's home by health workers and then the sample will be sent to the Immunology Section);&#xD;
      T2 = after 14 days from diagnosis similar to that described for the previous collection. All&#xD;
      the samples will be collected into vacutainer serum separator tubes (SST, Vacutainer®, Becton&#xD;
      &amp; Dickinson). In some cases, patients admitted to ICU also receive BAL (bronchiole-alveolar&#xD;
      lavage) or tracheal / mini BAL aspiration. The BAL will be collected whenever it is an&#xD;
      integral part of the patient's diagnostic path or within thirty minutes of the patient's&#xD;
      confirmed death. In all cases, together with the BAL it will also be necessary to collect a&#xD;
      test tube of blood, independently and in addition to the samples described above.&#xD;
&#xD;
      These samples will be analyzed for: a) a complete immune phenotype by flow cytometry; b) a&#xD;
      panel of soluble factots (i.e. cytokines), c) a deep immunological composition by single-cell&#xD;
      RNA sequencing, d) presence of viral sequences.&#xD;
&#xD;
      Analysis will be performed at Immunology Section (a,b,c) and Microbiology and Virology&#xD;
      Section of Hospital Trust of Verona.&#xD;
&#xD;
      Immunophenotype analysis on whole peripheral blood will be performed according to a standard&#xD;
      no-wash procedure, using a PrepPlus™ 2 workstation (Beckman Coulter Inc., Brea, CA, USA).&#xD;
      Blood samples will be incubated with antibodies (Beckman Coulter Inc., Brea, CA, USA) for 15&#xD;
      min at 20°C. Following is the list of antibodies used:&#xD;
&#xD;
        -  FITC-conjugated CD57 (clone NC1), PE-conjugated CD45RA (ALB11), ECD-conjugated CD8&#xD;
           (SFCI21Thy2D3), PC5.5-conjugated CD56 (N901), PC7-conjugated CD4 (SFCI12T4D11),&#xD;
           APC-conjugated CD27 (1A4CD27), APC A700-conjugated CD45 (J33), APC A750-conjugated CD3&#xD;
           (UCHT1), PB-conjugated CD16 (3G8), KR OR-conjugated CD19 (J3-119)&#xD;
&#xD;
        -  FITC-conjugated CD57 (clone NC1), PE-conjugated HLA-DR (Immu-357), ECD-conjugated CD8&#xD;
           (SFCI21Thy2D3), PC5.5-conjugated CD38 (LS198-4-3), PC7-conjugated CD4 (clone&#xD;
           SFCI12T4D11), APC-conjugated CD14 (RMO52), APC A700-conjugated CD45 (J33), APC&#xD;
           A750-conjugated CD3 (UCHT1), PB-conjugated CD16 (3G8), KR OR-conjugated CD19 (J3-119)&#xD;
&#xD;
        -  PE-conjugated IgD (IA6-2), ECD-conjugated CD3 (UCHT1), PC5.5-conjugated CD27 (1A4CD27),&#xD;
           PC7-conjugated CD20 (clone B9E9), APC-conjugated IgM (SA-DA4), APC A700-conjugated CD45&#xD;
           (J33), APC A750-conjugated CD38 (LS198-4-3), PB-conjugated CD21 (BL13), KR OR-conjugated&#xD;
           CD19 (J3-119) Red blood cell lysis and cell fixation will be performed using TQ-Prep&#xD;
           workstation and ImmunoPrep reagent system (Beckman Coulter Inc., Brea, CA, USA). Samples&#xD;
           will be acquired with Navios and analyzed with Navios software (Beckman Coulter Inc.,&#xD;
           Brea, CA, USA).&#xD;
&#xD;
      For detection of cytokines, sera of COVID-19 patients were tested for GM-CSF, G-CSF, M-CSF,&#xD;
      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,&#xD;
      IL-17F, IL-17E, IL-21, IL-22, IL-23, IL-27, IL-28A, IL-31, IL-33, IL-34, MIP-3α/CCL20, CCL2,&#xD;
      TNF-α, TNF-β, TGFβ production by Human ProcartaPlex™ Panel 1 multiplex (ThermoFisher&#xD;
      Scientific, Waltham, MA, USA).&#xD;
&#xD;
      BAL cells and circulating immune cells will be purified and cell counts will be adjusted&#xD;
      following 10xgenomics protocol. Briefly, the cell suspension was super-loaded with 50,000&#xD;
      cells, in the ChromiumTM Controller for partitioning single cells into nanoliter-scale Gel&#xD;
      Bead-In-Emulsions (GEMs). Single Cell 3' reagent kit v3.1 will be used for reverse&#xD;
      transcription, cDNA amplification and library construction of the gene expression libraries&#xD;
      (10x Genomics) following the detailed protocol provided by 10xGenomics. Hashtag libraries&#xD;
      will be prepared according to the cell hashing protocol for 10x Single Cell 3' Reagent Kit&#xD;
      v3.1. Biometra Trio Thermal Cycler will be used for amplification and incubation steps&#xD;
      (Analytik Jena). Libraries will be quantified by QubitTM 2.0 Fluorometer (ThermoFisher) and&#xD;
      quality will be checked using 2100 Bioanalyzer with High Sensitivity DNA kit (Agilent).&#xD;
      Sequencing will be performed in paired-end mode with a S1 and S2 flow cell (2 × 50 cycles&#xD;
      kit) using NovaSeq 6000 sequencer (Illumina).&#xD;
&#xD;
      The short history of this disease outbreak makes robust statistical power testing difficult.&#xD;
      However, for analysis during the treatment the investigators will consider only those&#xD;
      patients who are still alive, for whom the clinical and laboratory data for at least two time&#xD;
      points are available. Analysis on the duration of hospitalization will be performed in all&#xD;
      the patients enrolled except for the ones who died. Quantitative variables will be expressed&#xD;
      as the median and interquartile range (IQR), qualitative ones as percentages. To perform&#xD;
      pairwise comparisons, significance of difference will be evaluated by the Mann-Witney U-test&#xD;
      for quantitative variables and Fisher's exact test for categorical variables. The Wilcoxon&#xD;
      matched-pairs signed-rank test will be used to test the equality of matched pairs of&#xD;
      observation. Multiple logistic-regression analysis will be performed to assess the&#xD;
      association between the treatment and mortality for known negative prognostic factor (age,&#xD;
      sex and D-dimer). The strength of the association will be expressed by the Hazard Ratio (HR)&#xD;
      with 95% confidence interval (CI). Statistical significance will set at p-value &lt;0.05, and&#xD;
      clinical analyses will be performed using statistical software STATA version 16.0 (StataCorp,&#xD;
      College Station, TX, USA). For statistical laboratory analyses the investigators will perform&#xD;
      paired comparisons by Student's t-test and one-way Anova test for repeated measures, using&#xD;
      Graph Pad Prism (San Diego, California, version 8.4.2).&#xD;
&#xD;
      All biological materials will be stored after anonymisation at of Medicine Department of&#xD;
      University and Hospital Trust of Verona for the duration of the study (8 months). After that,&#xD;
      all biological materials will be transferred at Biobanca of University and Hospital Trust of&#xD;
      Verona. All patients data will be only accessed by researchers involved in the study with a&#xD;
      personnel account. All patients are anonymzed during immunological analysis. All data are&#xD;
      protected by software and database of University and Hospital Trust of Verona.&#xD;
&#xD;
      The ownership of the data will only be referred to the Principal Investigator (PI) of the&#xD;
      project.&#xD;
&#xD;
      TIMELINE OF THE PROJECT Duration of specimen collection: 6 months. This period was estimated&#xD;
      on the need to treat patients with COVID-19 immediately Expected duration for the analysis of&#xD;
      the data obtained: 2 months. This period has been estimated from the investigators'&#xD;
      experience and includes both the analysis of the samples and the statistical analysis of the&#xD;
      data obtained.&#xD;
&#xD;
      Total duration of the project: 8 months&#xD;
    </textblock>
  </detailed_description>
  <overall_status>Unknown status</overall_status>
  <last_known_status>Recruiting</last_known_status>
  <start_date type="Actual">March 26, 2020</start_date>
  <completion_date type="Anticipated">November 26, 2020</completion_date>
  <primary_completion_date type="Actual">June 10, 2020</primary_completion_date>
  <study_type>Observational</study_type>
  <has_expanded_access>No</has_expanded_access>
  <study_design_info>
    <observational_model>Cohort</observational_model>
    <time_perspective>Prospective</time_perspective>
  </study_design_info>
  <primary_outcome>
    <measure>COVID-19 associated immune disorder</measure>
    <time_frame>24 hours</time_frame>
    <description>Enumeration of circulating cell subsets by flow cytometry [Cell count/µl]</description>
  </primary_outcome>
  <primary_outcome>
    <measure>COVID-19 associated inflammation</measure>
    <time_frame>48 hours</time_frame>
    <description>Quantification of plasma levels of different solubles factors (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) [pg/ml]</description>
  </primary_outcome>
  <primary_outcome>
    <measure>Oxygenation</measure>
    <time_frame>24 hours</time_frame>
    <description>Ratio of arterial oxygen tension (mmHg) to fraction of inspired oxygen (PaO2/FiO2)</description>
  </primary_outcome>
  <primary_outcome>
    <measure>Diagnostic of COVID disease composite</measure>
    <time_frame>On admission of hospital</time_frame>
    <description>SARS-CoV-2 infection will be tested by PCR using nasopharyngeal swab</description>
  </primary_outcome>
  <secondary_outcome>
    <measure>Changes at the cytokine pattern</measure>
    <time_frame>14 Days</time_frame>
    <description>Quantification of plasma levels of different solubles factors (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) [pg/ml]</description>
  </secondary_outcome>
  <secondary_outcome>
    <measure>Changes at circulating immune cell composition</measure>
    <time_frame>14 Days</time_frame>
    <description>Enumeration of circulating cell subsets by flow cytometry [Cell count/µl]</description>
  </secondary_outcome>
  <secondary_outcome>
    <measure>Intensive Care Unit Admission</measure>
    <time_frame>Day 7-14</time_frame>
    <description>Proportion of patients with Intensive Care Unit Admission requirement</description>
  </secondary_outcome>
  <secondary_outcome>
    <measure>Length of hospital stay</measure>
    <time_frame>Day 7-14</time_frame>
    <description>Days of Hospitalization</description>
  </secondary_outcome>
  <secondary_outcome>
    <measure>Clinical Status</measure>
    <time_frame>Day 7-14</time_frame>
    <description>Clinical status assessed according to the World Health Organization guideline</description>
  </secondary_outcome>
  <secondary_outcome>
    <measure>Mortality</measure>
    <time_frame>Day 7-14</time_frame>
    <description>Proportion of death patients at days</description>
  </secondary_outcome>
  <number_of_groups>3</number_of_groups>
  <enrollment type="Anticipated">80</enrollment>
  <condition>Allergy and Immunology</condition>
  <condition>Infection Viral</condition>
  <arm_group>
    <arm_group_label>Mild disease</arm_group_label>
    <description>COVID-19 hospitalized patients</description>
  </arm_group>
  <arm_group>
    <arm_group_label>Severe disease</arm_group_label>
    <description>COVID-19 hospitalized patients in intensive care unit</description>
  </arm_group>
  <arm_group>
    <arm_group_label>paucisymptomatic syndrome</arm_group_label>
    <description>Mild symptomatic patients in home quarantine</description>
  </arm_group>
  <intervention>
    <intervention_type>Drug</intervention_type>
    <intervention_name>COVID-19 treatment</intervention_name>
    <description>COVID-19 affected patients treated with hydroxicloroquine or antiviral therapy (lopinavir/ritonavir) as single agents or in combination (hydroxicloroquine plus antiviral therapy) according to clinical features. Other concomitant treatments (i.e. off-label therapy) will be provided at the discretion of clinicians. Supportive therapy, such as antibiotic prophylaxis and anticoagulant treatment, will be provided at the discretion of the clinicians.</description>
    <arm_group_label>Mild disease</arm_group_label>
    <arm_group_label>Severe disease</arm_group_label>
    <arm_group_label>paucisymptomatic syndrome</arm_group_label>
  </intervention>
  <eligibility>
    <study_pop>
      <textblock>
        Hospitalized adults affected by SARS-CoV-2.&#xD;
      </textblock>
    </study_pop>
    <sampling_method>Non-Probability Sample</sampling_method>
    <criteria>
      <textblock>
        Inclusion Criteria:&#xD;
&#xD;
          -  Hospitalized adults affected by SARS-CoV-2. These patients can be enrolled also for&#xD;
             other clinical studies such as off-label or compassionate treatments.&#xD;
&#xD;
        Exclusion Criteria:&#xD;
&#xD;
          -  Patients that withdraw informed consent&#xD;
      </textblock>
    </criteria>
    <gender>All</gender>
    <minimum_age>18 Years</minimum_age>
    <maximum_age>90 Years</maximum_age>
    <healthy_volunteers>No</healthy_volunteers>
  </eligibility>
  <overall_official>
    <last_name>VINCENZO BRONTE, MD</last_name>
    <role>Principal Investigator</role>
    <affiliation>University and Hospital Trust of Verona</affiliation>
  </overall_official>
  <overall_contact>
    <last_name>STEFANO UGEL</last_name>
    <phone>0458126451</phone>
    <phone_ext>0039</phone_ext>
    <email>stefano.ugel@univr.it</email>
  </overall_contact>
  <location>
    <facility>
      <name>Azienda Ospedaliera Universitaria Integrata Verona</name>
      <address>
        <city>Verona</city>
        <zip>37126</zip>
        <country>Italy</country>
      </address>
    </facility>
    <status>Recruiting</status>
    <contact>
      <last_name>STEFANO UGEL</last_name>
      <phone>0458126451</phone>
      <phone_ext>0039</phone_ext>
      <email>stefano.ugel@univr.it</email>
    </contact>
    <investigator>
      <last_name>VINCENZO BRONTE, MD</last_name>
      <role>Principal Investigator</role>
    </investigator>
  </location>
  <location_countries>
    <country>Italy</country>
  </location_countries>
  <reference>
    <citation>Moore JB, June CH. Cytokine release syndrome in severe COVID-19. Science. 2020 May 1;368(6490):473-474. doi: 10.1126/science.abb8925. Epub 2020 Apr 17. No abstract available.</citation>
    <PMID>32303591</PMID>
  </reference>
  <reference>
    <citation>Mehta P, McAuley DF, Brown M, Sanchez E, Tattersall RS, Manson JJ; HLH Across Speciality Collaboration, UK. COVID-19: consider cytokine storm syndromes and immunosuppression. Lancet. 2020 Mar 28;395(10229):1033-1034. doi: 10.1016/S0140-6736(20)30628-0. Epub 2020 Mar 16. No abstract available.</citation>
    <PMID>32192578</PMID>
  </reference>
  <reference>
    <citation>Tay MZ, Poh CM, Renia L, MacAry PA, Ng LFP. The trinity of COVID-19: immunity, inflammation and intervention. Nat Rev Immunol. 2020 Jun;20(6):363-374. doi: 10.1038/s41577-020-0311-8. Epub 2020 Apr 28.</citation>
    <PMID>32346093</PMID>
  </reference>
  <reference>
    <citation>Wen W, Su W, Tang H, Le W, Zhang X, Zheng Y, Liu X, Xie L, Li J, Ye J, Dong L, Cui X, Miao Y, Wang D, Dong J, Xiao C, Chen W, Wang H. Immune cell profiling of COVID-19 patients in the recovery stage by single-cell sequencing. Cell Discov. 2020 May 4;6:31. doi: 10.1038/s41421-020-0168-9. eCollection 2020. Erratum In: Cell Discov. 2020 Jun 20;6:41.</citation>
    <PMID>32377375</PMID>
  </reference>
  <reference>
    <citation>Merad M, Martin JC. Pathological inflammation in patients with COVID-19: a key role for monocytes and macrophages. Nat Rev Immunol. 2020 Jun;20(6):355-362. doi: 10.1038/s41577-020-0331-4. Epub 2020 May 6. Erratum In: Nat Rev Immunol. 2020 Jun 2;:</citation>
    <PMID>32376901</PMID>
  </reference>
  <reference>
    <citation>Zhou F, Yu T, Du R, Fan G, Liu Y, Liu Z, Xiang J, Wang Y, Song B, Gu X, Guan L, Wei Y, Li H, Wu X, Xu J, Tu S, Zhang Y, Chen H, Cao B. Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study. Lancet. 2020 Mar 28;395(10229):1054-1062. doi: 10.1016/S0140-6736(20)30566-3. Epub 2020 Mar 11. Erratum In: Lancet. 2020 Mar 28;395(10229):1038. Lancet. 2020 Mar 28;395(10229):1038.</citation>
    <PMID>32171076</PMID>
  </reference>
  <verification_date>March 2020</verification_date>
  <study_first_submitted>June 16, 2020</study_first_submitted>
  <study_first_submitted_qc>June 18, 2020</study_first_submitted_qc>
  <study_first_posted type="Actual">June 19, 2020</study_first_posted>
  <last_update_submitted>June 18, 2020</last_update_submitted>
  <last_update_submitted_qc>June 18, 2020</last_update_submitted_qc>
  <last_update_posted type="Actual">June 22, 2020</last_update_posted>
  <responsible_party>
    <responsible_party_type>Sponsor</responsible_party_type>
  </responsible_party>
  <keyword>COVID-19</keyword>
  <keyword>IMMUNE SYSTEM</keyword>
  <keyword>INNATE IMMUNITY</keyword>
  <keyword>ADAPTIVE IMMUNITY</keyword>
  <condition_browse>
    <!-- CAUTION:  The following MeSH terms are assigned with an imperfect algorithm            -->
    <mesh_term>COVID-19</mesh_term>
    <mesh_term>Virus Diseases</mesh_term>
  </condition_browse>
  <pending_results>
    <submitted>January 31, 2023</submitted>
  </pending_results>
  <!-- Results have not yet been posted for this study                                          -->
</clinical_study>


download date: ClinicalTrials.gov processed this data on September 20, 2023
link text: Link to the current ClinicalTrials.gov record.
url: https://clinicaltrials.gov/ct2/show/NCT04438629
org study id: IMMUNOVID 2590CESC
nct id: NCT04438629
lead sponsor: 
is fda regulated drug: No
is fda regulated device: No
The aim of the project is to evaluate the immunological features of COVID-19 patients.
      Patients are recruited without any pharmacological treatments restriction. The number of
      samples is estimated on the basis of feasibility, that means on the maximum number of
      patients with COVID-19, who are expected to be able to be enrolled by the units involved.
      Based on the investigators' experience, gained in the onco-immunological field, considering
      the time and economic resources available, the investigators expect to enroll at least 80
      patients.
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
observational model: Cohort
time perspective: Prospective
measure: COVID-19 associated immune disorder
time frame: 24 hours
description: Enumeration of circulating cell subsets by flow cytometry [Cell count/µl]
measure: COVID-19 associated inflammation
time frame: 48 hours
description: Quantification of plasma levels of different solubles factors (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) [pg/ml]
measure: Oxygenation
time frame: 24 hours
description: Ratio of arterial oxygen tension (mmHg) to fraction of inspired oxygen (PaO2/FiO2)
measure: Diagnostic of COVID disease composite
time frame: On admission of hospital
description: SARS-CoV-2 infection will be tested by PCR using nasopharyngeal swab
measure: Changes at the cytokine pattern
time frame: 14 Days
description: Quantification of plasma levels of different solubles factors (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) [pg/ml]
measure: Changes at circulating immune cell composition
time frame: 14 Days
description: Enumeration of circulating cell subsets by flow cytometry [Cell count/µl]
measure: Intensive Care Unit Admission
time frame: Day 7-14
description: Proportion of patients with Intensive Care Unit Admission requirement
measure: Length of hospital stay
time frame: Day 7-14
description: Days of Hospitalization
measure: Clinical Status
time frame: Day 7-14
description: Clinical status assessed according to the World Health Organization guideline
measure: Mortality
time frame: Day 7-14
description: Proportion of death patients at days
arm group label: Mild disease
description: COVID-19 hospitalized patients
arm group label: Severe disease
description: COVID-19 hospitalized patients in intensive care unit
arm group label: paucisymptomatic syndrome
description: Mild symptomatic patients in home quarantine
intervention type: Drug
intervention name: COVID-19 treatment
description: COVID-19 affected patients treated with hydroxicloroquine or antiviral therapy (lopinavir/ritonavir) as single agents or in combination (hydroxicloroquine plus antiviral therapy) according to clinical features. Other concomitant treatments (i.e. off-label therapy) will be provided at the discretion of clinicians. Supportive therapy, such as antibiotic prophylaxis and anticoagulant treatment, will be provided at the discretion of the clinicians.
arm group label: Mild disease
arm group label: Severe disease
arm group label: paucisymptomatic syndrome
study pop: 
sampling method: Non-Probability Sample
criteria: 
gender: All
minimum age: 18 Years
maximum age: 90 Years
healthy volunteers: No
last name: VINCENZO BRONTE, MD
role: Principal Investigator
affiliation: University and Hospital Trust of Verona
last name: STEFANO UGEL
phone: 0458126451
phone ext: 0039
email: stefano.ugel@univr.it
facility: 
status: Recruiting
contact: 
investigator: 
country: Italy
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PMID: 32303591
citation: Mehta P, McAuley DF, Brown M, Sanchez E, Tattersall RS, Manson JJ; HLH Across Speciality Collaboration, UK. COVID-19: consider cytokine storm syndromes and immunosuppression. Lancet. 2020 Mar 28;395(10229):1033-1034. doi: 10.1016/S0140-6736(20)30628-0. Epub 2020 Mar 16. No abstract available.
PMID: 32192578
citation: Tay MZ, Poh CM, Renia L, MacAry PA, Ng LFP. The trinity of COVID-19: immunity, inflammation and intervention. Nat Rev Immunol. 2020 Jun;20(6):363-374. doi: 10.1038/s41577-020-0311-8. Epub 2020 Apr 28.
PMID: 32346093
citation: Wen W, Su W, Tang H, Le W, Zhang X, Zheng Y, Liu X, Xie L, Li J, Ye J, Dong L, Cui X, Miao Y, Wang D, Dong J, Xiao C, Chen W, Wang H. Immune cell profiling of COVID-19 patients in the recovery stage by single-cell sequencing. Cell Discov. 2020 May 4;6:31. doi: 10.1038/s41421-020-0168-9. eCollection 2020. Erratum In: Cell Discov. 2020 Jun 20;6:41.
PMID: 32377375
citation: Merad M, Martin JC. Pathological inflammation in patients with COVID-19: a key role for monocytes and macrophages. Nat Rev Immunol. 2020 Jun;20(6):355-362. doi: 10.1038/s41577-020-0331-4. Epub 2020 May 6. Erratum In: Nat Rev Immunol. 2020 Jun 2;:
PMID: 32376901
citation: Zhou F, Yu T, Du R, Fan G, Liu Y, Liu Z, Xiang J, Wang Y, Song B, Gu X, Guan L, Wei Y, Li H, Wu X, Xu J, Tu S, Zhang Y, Chen H, Cao B. Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study. Lancet. 2020 Mar 28;395(10229):1054-1062. doi: 10.1016/S0140-6736(20)30566-3. Epub 2020 Mar 11. Erratum In: Lancet. 2020 Mar 28;395(10229):1038. Lancet. 2020 Mar 28;395(10229):1038.
PMID: 32171076
responsible party type: Sponsor
mesh term: COVID-19
mesh term: Virus Diseases
submitted: January 31, 2023