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Role of S309-CAR-NK in Neutralizing SARS-CoV2 

Rationale:

In SARS-CoV-2, the variant’s properties influence the effectiveness of public health interventions, some even increase their transmissibility. Other properties of the variants are that they can also change the virus’s antigenicity (the ability of an antigen to induce an immunological response when it encounters the human body), avoid the immunity caused by previous infection/vaccination, and evade the immune response. All these characteristics increase the virus’s severity. For example, a variant of concern B.1.1.7 first identified in the UK was dominant in London and southeast England. It had the characteristic of being 50% more transmissible than previous variants. Omicron, characterized by 17 mutational changes, increased the number of cases dramatically by having a short generation time for transmission with a 59% higher transmissibility and 45% higher mortality rate, with a substantial antigenic change. The Omicron variant has efficient growth in the upper airway, and a bigger pool of children susceptible compared to adults, resulting in enhanced SARS-CoV2 evolution and declined vaccine efficacy. In the virus, there are two functional regions: S1 which consists of the receptor binding domain (RBD), and S2. S1 is responsible for the ability of the virus to bind to human cells to facilitate infection. Antigens against RBD have been seen as being able to stop the virus from binding to infect human cells. The spike proteins bind to the ACE2 receptors at the surface of healthy cells, then S2 is to fuse with the cell membrane of the healthy cell. During binding, S1 undergoes conformational rearrangement between up and down conformation states. The down conformation depicts how it hides the receptor binding domain (closed), and up exposes it but temporarily destabilizes the protein subunit (open). Antibodies for the coronavirus recognize and bind the spike protein to prevent the virus from infecting a healthy host cell. Neutralizing antibodies such as S309, isolated from a SARS patient, could diffuse not only this specific viral strain but also offshoots that occur because of natural mutations in the virus. S309 targets the RBD epitome which is mainly VH (Immunoglobin heavy chain variable region) mediated. 

NK cells, also known as Natural Killer Cells, are a type of immune white blood cell that has granules that can destroy tumors and other virus-infected cells. Mature NK cells constitute about 10-15 % of circulating lymphocytes and are found in the spleen, bone marrow, liver (pit cells), lung, intestinal mucosa, uterus, and small amounts of lymph nodes. They can travel in and out of tissue and bloodstream and play a key role in the host’s immune defense against pathogens, preventing the establishment of infection and the viral spreading through the body. Their functions include releasing cytotoxic granules which punch holes in the target cell’s membrane by binding directly to phospholipids, creating pores, then releasing some molecules that get inside the target cell causing them to undergo apoptosis (type of program cell death). NK cells are affected in the bloodstream by SARS-CoV2, which SARS-CoV2 interrupts equations of immune responses, disrupting cytolytic antiviral effects of NK cells, and inducing a “cytokine storm” by activating infected immune cells. Adoptive transfer of proper cytolytic potentiated and lowest capacity of cytokine released NK cells and CAR-NK cells may be effective for this problem. 

In a process like blood donation where a patient’s T cells are collected to reprogram T cells to produce special receptors on their surface known as chimeric antigen receptors (CAR) to recognize specific antigens on the cancer cells and destroy them. After the CAR-T cells are grown in a lab, they are infused back into patients. CAR-NK cells are a safer alternative to CAR-T cell therapy. Their advantages include less antigen loss relapse, minimal on-target, off-tumor toxicity, and antibody-dependent cellular cytotoxicity (ADCC). By having various activating and inhibitory receptors, it allows the CAR-NK cells to have stronger and less off-target reactions. Not only that, CAR-NK cells have a low risk of grafts versus host disease (GvHD), cytokine release syndrome (CRS), and immune effector cell-associated neurotoxicity syndrome (ICANS), where these side effects are often seen in CAR-T therapy due to allogeneic donors. This makes CAR-NK cells good candidates for “off-the-shelf” cellular immunotherapy treatment development because no evidence of GvHD is found in almost all clinical studies of NK cells. They release IFN-γ and GM-CSF that causes fewer toxic complications and reduces the occurrence of cytokine storms, making it a safe way of cell-based immunotherapy.

Given this information about SARS-CoV-2, their variants’ impact, especially Omicron, on public health, and the physical structure of the current variant of the SARS-CoV-2 virus, I’ve learned about the critical position the public is in. With this information along with learning about Natural Killer cells’ role in the immune system and S309-CAR-NK’s part in immunotherapy for this virus, I hypothesize that S309-CAR-NK cells can bind to Omicron subvariant XBB.1.5 Spike protein; therefore, neutralizing the pseudoviral SARS-CoV2 XBB.1.5 particles in vitro and can be used as a potential therapeutic for immunocompromised COVID patients. I will design these experiments as followed to test my hypothesis:

Research Questions: 

Do S309-CAR-NK cells express the S309 receptor?

Do S309-CAR-NK cells recognize the spike protein of Omicron variant XBB.1.5?

Do S309-CAR-NK cells bind to the spike protein of Omicron variant XBB.1.5?

Procedure:

1. Determine the total expression of S309-CAR-NK cells using western blot:
   1. Add SDS and beta-mercaptoethanol into the protein and put protein into incubator to boil for 10 minutes 
   2. Quantify protein concentrations using BCA kit.
   3. Load 20 μg of protein per lane. Using the micropipette (15 microliters) carefully add the protein sample and loading dye into the wells, using a buffer in between the chains. 
   4. Fill the apparatus with running buffer.
   5. Attach the box's lid with the power supply and turn on the desired voltage. Watch to see if bubbles appear to be rising. Run gel until blue line reaches the bottom.
   6. Proteins now negatively charged because they are coated by the SDS. They will move from negative (top) to the positive terminal (bottom). 
   7. Proteins stacked on top of the resolving gel and they resolve according to the function of their molecular weight

WESTERN BLOT

8. While waiting for the gel, soak the sponges needed for western blotting in the transfer buffer.
9. Cut pieces of the PVDF membrane and place them in a box to be treated with methanol and transfer buffer. 
10. Remove the gel once it’s done.
11. Place sponge on the clear side of the chamber, then on top, add a piece of filter paper, then the gel, membrane, a piece of filter paper, and the sponge. 
12. Close the chamber with the “sandwich” inside. 
13. Place the chamber into a box and cover with transfer buffer. Add transfer buffer to the outer box, close the lid, and turn on the voltage at 100V for 1 hour in the cold room. Once it’s done, disassemble the module and place the membrane in a box. 
14. Pour the 5% milk blocking buffer over the membrane and place it on the rocker for 1 hour at room temperature.
15. Add anti-CD3z primary antibodies at 1:1000 dilution and incubate at room temperature for 1 hour.
16. Wash blot 5 times, 5 minutes each with PBST buffer.
17. Add 1:2000 dilution anti-rabbit secondary antibody then incubate for 1 hour at room temperature. 
18. Wash blot 5 times, 5 minutes each with PBST buffer.
19. Add ECL then expose the membrane using a Chemidoc
20. Strip membrane for 15 minutes at room temperature then wash blot 2 times, 5 minutes each with PBST buffer.
21. Add conjugated anti-β-actin (housekeeping protein) at 1:10,000 dilution and incubate at room temperature for 1 hour.
22. Wash blot 5 times, 5 minutes each with PBST buffer.
23. Add ECL then expose the membrane using a Chemidoc
24. Determine the surface expression of S309-CAR-NK cells using flow cytometry:
    1. Parental non-modified NK cells will be used as negative control
    2. All centrifuging in protocol should be completed at 1250-1500 RPM or 300-500 g for five minutes.
    3. Label FACS tubes to be arranged in tube rack. 
    4. Harvest cells and on last wash step resuspend the cells at 1 million cells/100 microliters of flow cytometry staining buffer (1x10^6/100μL)
    5. Aliquot 100 microliters into each facx tube 
    6. Add conjugated antibody concentrated at 5-10 microliters per million cells ( 5-10μL/10^6) and vortex. 
       1. CD56-PE-Cy7 for NK cells
       2. CD3-PE for T cells
       3. hIgG-APC for CAR marker
    7. Incubate cells for 30 minuets in the dark on ice.
    8. Wash cells in 2 milliliters of flow cytometry staining buffer to remove unbound antibody.
    9. Centrifuge the cells 
    10. Vortex to resuspend the cells. 
    11. Add 2 milliliters of flow cytometry staining buffer and repeat centrifugation. 
    2. Resuspend cells
       1. In 200-400 microliters of flow cytometry staining buffer for flow cytometry analysis. 
25. Investigate whether S309-CAR-NK cells bind to Omicron subvariant XBB.1.5 Spike protein 
    1. Parental non-modified NK cells will be used as negative control
    2. All centrifuging in protocol should be completed at 1250-1500 RPM or 300-500 GS for five minutes.
    3. Label facs tubes to be arranged in tube rack. 
    4. Harvest cells and on last wash step resuspend the cells at 1 million cells/100 microliters of flow cytometry staining buffer (1x10^6/100μL)
    5. Aliquot 100 microliters into each facx tube 
    6. Add 1 μg of Spike omicron protein. Vortex. Incubate on ice for 30 minutes.
    7. Wash cells with facs buffer.
    8. Add conjugated antibody concentrated at 5-10 microliters per million cells ( 5-10μL/10^6) and vortex. 
       1. CD56-PE-Cy7 for NK cells
       2. CD3-PE for T cells
       3. hIgG-APC for CAR markers
       4. anti-RBD to detect spike protein binding to CAR-NK cells
    9. Incubate cells for 30 minuets in the dark at room temperature.
    10. Wash cells in 2 milliliters of flow cytometry staining buffer to remove unbound antibody.
    11. Centrifuge the cells 
    12. Vortex to resuspend the cells. 
    13. Add 2 milliliters of flow cytometry staining buffer and repeat centrifugation. 
    2. Resuspend cells
       1. In 200-400 microliters of flow cytometry staining buffer for flow cytometry analysis. 

Risk and Safety:

Biohazard chemicals include the use of sodium dodecyl sulfate (SDS), methanol, and beta-mercaptoethanol. To mitigate the risks of exposure, these chemicals will be handled inside a fume hood. As NK92MI is a cancer cell line and is derived from a cancer patient, additional precautions will be needed such as staining cells for flow cytometry in the hood. Personal protective equipment (PPE) including goggles, gloves, a lab coat, and closed-toe shoes will be always worn during experiments. The use of some equipment, such as a flow cytometer and heat block (used to denature proteins for the SDS-PAGE) may be dangerous and will be done by a supervised adult. All activities performed in the lab will be closely monitored by a supervisor adult.