SARS-CoV-2 Spike Expression at the Surface of Infected Primary Human Airway Epithelial Cells
, Shang Yu Gong 1,4, Romain Gasser 1,3, Annemarie Laumaea 1,3, Sai Priya Anand 1,4, Anik Privé 1, Catherine Bourassa 1, Halima Medjahed 1, Jérémie Prévost 1,3, Hugues Charest 5, Jonathan Richard 1,3, Emmanuelle Brochiero 1,2 and Andrés Finzi 1,3,4,*
Round 1
Reviewer 1 Report
Thank you for allowing me to review the article titled “SARS-CoV-2 Spike expression at the surface of infected primary human airway epithelial cells”. Currently, the research on RBD-targeted neutralizing antibodies has been very perfect, but there is a lack of systematic analysis of functional antibodies with antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP). For this kind of research, the primary task is to build an effective ADCC (ADCP) detection model, so the quantification of S protein in infected cells is the primary factor for analysis. This study found that the SARS-CoV-2 Spike is abundantly expressed at the surface of infected human airway epithelial cells and revealed correlation between spike recognition at the surface of infected human airway epithelial cells strongly and its detection with commonly used serological assay such as its detection at the surface of transfected or transduced cells, pseudoviral neutralization as well as with Fc-mediated effector functions such as ADCC, measured in a cohort of SARS-CoV-2 naïve vaccinated and previously-infected individuals. These findings play an important role in research of functional antibodies with ADCC or ADCP against SARS-CoV-2.
Author Response
We thank reviewer #1 for his/her very positive assessment of our manuscript
Reviewer 2 Report
In the study of Ding et al. entitled “SARS-CoV-2 Spike expression at the surface of infected primary human airway epithelial cells,” the authors developed a FACS-based assay combining the cell-surface detection of the Spike and the intracellular detection of the nucleocapsid (N) in primary human airway epithelial cells (pAECs) infected with authentic SARS-CoV-2. For this, pAECs were infected with SARS-CoV-2 D614G or Alpha variants. This assay allows the distinction between infected (N+) versus uninfected (N-) cells. The authors observed robust expression of SARS-CoV-2 Spike at the surface of infected pAECs using the conformational-independent anti-S2 antibody. Infected cells were also readily recognized by plasma from convalescent and vaccinated individuals and correlated with several serological assays. This suggests that the antigenicity of Spike present at the surface of infected primary cells is maintained in serological assays involving the expression of the native full-length Spike. The study is important since many works express the spike protein in vitro systems to assess antigenicity against patient sera, including tests against the spike of new variants, such as Omicron. Therefore, the study should be accepted after minor revisions.
Minor revisions:
1) Page 1, abstract: Change the abstract as above.
2) Page 2, line 58: Remove “viruses”. The acronym SARS-CoV-2 already contains the term "virus": Severe Acute Respiratory Syndrome Coronavirus 2;
3) The study sample number is very small [eight convalescent individuals 6- and 11-weeks post symptoms onset (PSO) and nine SARS-CoV-2 naïve individuals obtained before (V0), 3 weeks (V1), and 12 weeks (V2) after the first dose of the BNT162b2 mRNA vaccine or 3 weeks after the second dose (V3)]. Could this not have influenced the study results?
4) Authors should expand the discussion of the manuscript, including seeking articles that used a larger sample size to make the result of the current study and the comparison of data more robust.
Author Response
Reviewer #2 (comments for authors):
In the study of Ding et al. entitled “SARS-CoV-2 Spike expression at the surface of infected primary human airway epithelial cells,” the authors developed a FACS-based assay combining the cell-surface detection of the Spike and the intracellular detection of the nucleocapsid (N) in primary human airway epithelial cells (pAECs) infected with authentic SARS-CoV-2. For this, pAECs were infected with SARS-CoV-2 D614G or Alpha variants. This assay allows the distinction between infected (N+) versus uninfected (N-) cells. The authors observed robust expression of SARS-CoV-2 Spike at the surface of infected pAECs using the conformational-independent anti-S2 antibody. Infected cells were also readily recognized by plasma from convalescent and vaccinated individuals and correlated with several serological assays. This suggests that the antigenicity of Spike present at the surface of infected primary cells is maintained in serological assays involving the expression of the native full-length Spike. The study is important since many works express the spike protein in vitro systems to assess antigenicity against patient sera, including tests against the spike of new variants, such as Omicron. Therefore, the study should be accepted after minor revisions.
Response: We thank reviewer #2 for his/her very positive assessment of our manuscript
Minor revisions:
1) Page 1, abstract: Change the abstract as above.
Response: done
2) Page 2, line 58: Remove “viruses”. The acronym SARS-CoV-2 already contains the term "virus": Severe Acute Respiratory Syndrome Coronavirus 2;
Response: done
3) The study sample number is very small [eight convalescent individuals 6- and 11-weeks post symptoms onset (PSO) and nine SARS-CoV-2 naïve individuals obtained before (V0), 3 weeks (V1), and 12 weeks (V2) after the first dose of the BNT162b2 mRNA vaccine or 3 weeks after the second dose (V3)]. Could this not have influenced the study results?
Response: we now acknowledge the small sample size as a limitation of the study in the revised manuscript.
4) Authors should expand the discussion of the manuscript, including seeking articles that used a larger sample size to make the result of the current study and the comparison of data more robust.
Response: as indicated in point #3, we acknowledge the small sample size of our study but as suggested by the reviewer, we now cite studies with a larger sample size.
We trust that, with these changes, the manuscript is now suitable for publication in Viruses.
Sincerely,
Andrés Finzi
Author Response File: 
Author Response.pdf
