Safety and Efficacy of a Non-replicating ChAdOx1 Vector Vaccine AZD1222 (COVISHIELD), for Prevention of COVID-19 in Patients With Liver Cirrhosis

• Study population: Group 1: Liver Cirrhosis Group 2: Control Group (Healthy controls)
• Study design: A Single Centre, Pilot Study,Adaptive Clinical Trial
• Study period: 1 year, Enrolment Period: Mar 2021 to Aug 2021
• Sample size with justification:

Study is being conducted as a pilot study. First 100 participants will be enrolled in both the groups. However a preliminary analysis will be done after enrollment of 50 participants in each group. Study is being conducted as a Adaptive Clinical Trial.

• Intervention: 2 IM doses (0.5 mL) of AZD1222(Covishield) at Day 0 and after 6-8 weeks of first dose (Day 42-56 days). This schedule will be followed for the first 100 participants, following which vaccination schedule as per the prevailing Government protocols shall be followed as a part of Adaptive Clinical Trial.
• Monitoring and assessment:

SARS CoV2 serum IgG Levels and neutralizing antibody titers:

The blood samples at baseline (pre first dose), day 28 (4 weeks/28 days after first dose and just before second dose), 8 weeks (or 4 weeks after second dose), 16 weeks (or 12 weeks after second dose), 28 weeks (or 24 weeks after second dose), 40 weeks (or 36 weeks after second dose) and 52 weeks ( or 48 weeks after second dose) Post vaccination will be screened for anti-SARS-CoV-2 IgG by commercially available chemiluminescent immunoassays (CLIA) SARS-CoV-2 IgG (Ortho Clinical Diagnostics, Raritan, NJ, USA)on VITROS ECi/ECiQ/3600 automated instrument. Results will be expressed as reactive with Serum/cut off ratio ≥ 1, and non-reactive with < 1 S/Co ratio.

All reactive samples will be further processed to measure the neutralizing antibody titer utilizing a surrogate virus neutralization test ELISA based commercial assay (sVNT)(GenScript cPass™ SARS-CoV-2 Neutralization Antibody Detection Kit) to look for neutralizing antibody response to the spike protein of the virus.

Real Time PCR for SARS CoV-2 infection:

A diagnosis of SARSCoV-2 infection will be confirmed by performance of Real Time reverse transcriptase polymerase chain reaction (RT-PCR) on the combined nasopharyngeal and oral swab collected in Viral transport medium and by detection of at least 2 specific gene targets of the SARS CoV-2 virus (E and RdRP genes).

Primary IFN-γ ELISPOT assay. The blood samples at baseline (pre first dose), day 28 (4 weeks/28 days after first dose and just before second dose), 8 weeks (or 4 weeks after second dose), 16 weeks (or 12 weeks after second dose), 28 weeks (or 24 weeks after second dose), 40 weeks (or 36 weeks after second dose) and 52 weeks ( or 48 weeks after second dose) will be collected. Primarily, cellular responses will be assessed using an ex-vivo interferon-γ enzyme-linked immunospot (ELISpot) assay to enumerate antigen-specific T cells. In brief, PBMCs will be isolated from whole blood by density gradient centrifugation, immediately cryopreserved in 90% fetal bovine serum and 10% dimethyl sulfoxide, and transferred to liquid nitrogen for storage.

IFN-γ ELISPOT assays will be performed using cryopreserved PBMC. Briefly, unfractionated PBMC will be thawed, resuspended at a concentration of 1 × 106 cells/ml in DMEM medium, and will be plated at 100 μl/well (1 × 105 cells/well) in 96-well, nitrocellulose-backed plates (Millipore Corp, Bedford, MA) previously coated with a PBS solution of anti-IFN-γ monoclonal antibody (1-D1K, 5 μg/ml; Mabtech Technologies, Nacka, Sweden). Along with negative control as, different cell stimuli (PMA; phytohemagglutinin 1 μg/ml and Ionomycin ) will be used in as positive controls., Plates will then incubated at 37°C for 40 h, and then will be harvested and read in ELISPOT reader. A vaccine-induced response will be defined as a ≥3-fold increase over the baseline response to the individual epitope peptide.

T cells Functionality:

The blood samples at baseline (pre first dose), day 28 (4 weeks/28 days after first dose and just before second dose), 8 weeks (or 4 weeks after second dose), 16 weeks (or 12 weeks after second dose), 28 weeks (or 24 weeks after second dose), 40 weeks (or 36 weeks after second dose) and 52 weeks ( or 48 weeks after second dose) Immune response measurements will be assessed using peripheral blood mononuclear cells (PBMC) obtained at all time points.

To investigate the CD4 and CD8 T cell functionality PBMCs will be cultured in 96 well plate in RPMI media containing 10% FBS, with or without PMA / Ionomycin (positive control) (PMA 2 ng/mL; ionomycin 1 μg/mL; Merck, Darmstadt, Germany) and 10 µg /ml LPS (Merck, Darmstadt, Germany ) at 37°C ,5% CO2 for 6-7 hours. After 1 hour of incubation, 1 μg/mL brefeldin A (BD Pharmingen, USA) will be added to all the wells. After incubation, cells will be surface stained for 25-30 minutes with anti-CD3FITC, anti-CD8BV421 and anti-CD4 PE/cyanine5.5 (Cy5) followed by 10 minutes permeabilization with 100 µL of permeabilising solution( BD Biosciences, USA) and will be washed twice with 500 µL 1x cytoperm. After washing, cells will be stained for intracellular expression of IL-2, IFN- γ and IL-17 with anti-IL2 APC, anti-IFN-γ PE, anti-IL-17 PE- Cy7 (BD Biosciences and Pharmingen, USA) and will be incubated for 25-30 minutes followed by washing with PBS and fixing the cells in 0.1 % PFA before acquiring on a BD Verse flow cytometer. Data will be analyzed using FlowJo software version 10.

Switching of monocyte Phenotype and functionality:

The blood samples at baseline (pre first dose), day 28 (4 weeks/28 days after first dose and just before second dose), 8 weeks (or 4 weeks after second dose), 16 weeks (or 12 weeks after second dose), 28 weeks (or 24 weeks after second dose), 40 weeks (or 36 weeks after second dose) and 52 weeks ( or 48 weeks after second dose) Vaccine will induce an increase in proinflammatory cytokine production, which is dependent on trained immunity in innate immune cells. We will characterize the phenotype and functionality of monocytes before vaccination and after vaccination using flowcytometric analysis.

Those who are fully immunized and still developing clinical Covid 19 infection will be grouped together and immunological profile will be studied.