Discussion worksheet

Journal Club is intended to be an informal learning space through which you can increase your understanding of peer-reviewed publications, learn about research design, practice talking about science, and discover new research areas. This worksheet is intended to guide preparations for your discussions.

Create your worksheet for this journal club ASAP and complete it at least the Wednesday before Thursday’s meeting. This exercise will facilitate your reading and analysis of the journal articles, and your discussion leaders and peers can provide feedbacks immediately after you post your comments in the worksheet

Rules:

Don't change the highest-level title, but you can remove any italic contents as you wish in the issue. Add your own subtitles if you want.
Feel free to add quotations using >, use[]() for links and just drop the images in the window to link figures.
- COVID-19 Vaccine & Therapeutics Tracker
- COVID-19 Vaccine & Therapeutics Tracker

Still confused?

Go look at this mindmap to grab some ideas
Here is an example discussion sheet #4
Check this out: Markdown cheatsheet

Main Research Question?

The authors wanted to see how SARS-CoV-2-sepcific antibody and T cell reactivities change over time (early and late timepoints/post-booster) among individuals who received AstraZeneca, Johnson&Johnson, Moderna, or Pfizer vaccine.

Methods & Data

Sample Collection:

The authors took samples from n = 423 participants who received 1 of 4 vaccines. They measured S-specific antibodies and IFN-y levels at an early and late time point for each.

Plaque Reduction Neutralization Test (PRNT):

Infect ARS-CoV-2 into Calu-3 cells (MOI = 0.01) and grew to P3
Stain plaques and calculate frequency of GFP-positive cells
PRNT50 = the dilution that would yield 50% reduction of plaques
Perform deep sequencing to confirm that P3 was equal to P1 and characterize any mutations

Flow Cytometry:

SARS-CoV-2-specific T cells were detected by flow cytometry
AIM = activation induced markers
CD3 activates CD4 and CD8 T cells
FSC-A and FSC-H = Area and Height
CCR7, CD45RA, Ox40, and CD137 stain for activated memory T cells
High expression of CD4/CD8, Spike results in 10-fold higher stimulation than the control DMSO condition

Authors' interpretation

Yes - I agree with the main finding that neutralizing antibodies are more important than T cells overall when mounting a SARS-CoV-2 immune response. Expanding the study would substantiate this finding.

Enhancement or next experiment?

The authors should expand the study! Some interesting possibilities are including different geographic areas (comparing vaccinated individuals across multiple continents or in rural/suburban/urban settings), increasing the age range to include children and older adults, and including immunocompromised or unvaccinated individuals.

confusion? Terms you don't understand?

Most of the paper was straightforward and easy to understand - clear figures and experimental design. For the flow cytometry portion, I had to look up the immunological markers and their significance. I'm still a bit unclear on the significance of FSC-H and FSC-A, which supposedly mean height and area, respectively. How does what's in the chart mean "singlet" and what is the significance of singlet?

Have your own opinions or critiques

My primary critique is the low sample size and the fact that they mostly included healthy individuals. However, I think they could've infected SARS-CoV-2 cells into another cell line and compared the results. What was their rationale for using Calu-3 cells?

I hereby confirm that I have:

[ ] I am still working on the worksheet
[X] I have finished the worksheet
[X] I have gone through the papers
[ ] Check your peer if they have similar ideas. If it is, plz add their issue number to your comments.

Some interesting possibilities are including different geographic areas (comparing vaccinated individuals across multiple continents or in rural/suburban/urban settings), increasing the age range to include children and older adults, and including immunocompromised or unvaccinated individuals.

Great points! But probably for next papers, if they can recruit/stratify them with enough sample size. Probably one thing they should explode and provide description is medical history, because during 6 months, a lot of things may happen (for example break through infection). Those should be reported. Did they report any cases that have breakthrough infection?

I had to look up the immunological markers and their significance. I'm still a bit unclear on the significance of FSC-H and FSC-A, which supposedly mean height and area, respectively. How does what's in the chart mean "singlet" and what is the significance of singlet?

FSC-A/FSC-H can be used for mainly two folds: (1) FSC can be roughly used for cell type differentiations. For example, APCs usually will be larger then T cells and it will be clearly to be seen in FSC. (2) The more important aspect is to remove doublets and gate only on "singlet". Why? Because if you include "doublet", they might increase marker staining intensity significantly. So those cells might be false positive when you used in the later gating procedure.

What was their rationale for using Calu-3 cells?

This is an excellent question! Explanation was given in page 2 of 12: Human airway Calu-3 cells were used for virus propagation and neutralization assays because SARS-CoV-2 enters these cells using the TMPRSS2-mediated entry pathway (36–39). This entry pathway is used in vivo and prevents adaptations in S, commonly observed in Vero cells.
See more in https://pubmed.ncbi.nlm.nih.gov/34960703/

gmhhope / fall-postbacc-journal-club-2022

DW-PBJ4-Megan #81