Global m6A RNA Methylation in SARS-CoV-2 Positive Nasopharyngeal Samples in a Mexican Population: A First Approximation Study

Round 1

Reviewer 1 Report

RNA modifications were reported to play important roles in RNA metabolism and functions both in the host and viruses. M6A modification of viral RNA has been widely studied. In the current study, the authors checked  whether m6A modification differs across 20 individuals infected with distinct SARS-CoV-2 variants, viral load or vaccination status by using a competitive immunoassay method from the patient samples of nasopharyngeal. The results showed that methylation was significantly lower in the delta- and omicron-positive samples compared to non-infected individuals. Further study revealed that the methylation levels was linked to the vaccination. The results are interesting. The following suggestions need to be considered before the full acceptance of the manscript.

1) m6A has been detected in the clinical sample in the previous literature. The authors can add the related information in the introduction part.

2) m6A modification has been reported by several groups. The difference among the papers need more details in the discussion part.

Author Response

We want to thank the four reviewers for their comments and the time to revise this manuscript. All major changes in the document are highlighted in yellow, however several minor changes (not highlighted) were made throughout the document, and several references were added.

Reviewer 1

1) m6A has been detected in the clinical sample in the previous literature. The authors can add the related information in the introduction part.

Thank you for the observation. Indeed m6A has been detected in clinical samples derived from peripheral blood and epithelial cells of bronchoalveolar lavage fluid from COVID-19 patients, but (at least to our knowledge) not in nasopharyngeal samples. We have included the following text in the Introduction section:

“Recent studies show that SARS-CoV-2 infection changes the host cell m6A methylome in vitro, promoting differential expression of host genes [13], and in vivo, altering m6A modification levels in lymphocytes from peripheral blood samples by increased expression of the m6A methyltransferase RNA-binding motif protein 15 (RBM15) [17], or decreased expression of METTL3 in epithelial cells of bronchoalveolar lavage fluid of COVID-19 patients [13]”.

2) m6A modification has been reported by several groups. The difference among the papers need more details in the discussion part.

Thank you for your comment. We included more detailed explanations regarding m6A levels and expression of the genes associated with the methylation machinery in COVID-19 samples.

We included this information in the Introduction:

“To date, only a few studies have probed for alterations in global m6A levels in a limited number of COVID-19 positive individuals (n = 2 – 20) [13, 17, 18]. These studies found increased levels of m6A in peripheral blood samples [17] or in epithelial cells of bronchoalveolar lavage fluid [13] in infected individuals relative to non-infected individuals that are associated with increased expression of RBM15, that encodes for a protein that facilitates m6A by guiding METTL3 to the target RNA sequence [17].”

We included this information in the Discussion:

“…previous studies have reported a higher m6A level in lung or peripheral blood from patients with moderate or severe COVID-19 disease compared to healthy individuals [13, 17, 18, 26]. Interestingly, in peripheral blood, these changes were associated with increased expression of RBM15 [17]. Moreover, Qiu et al. [26] proposed a predictive “m6A score” to quantify and model the m6A pattern in blood leukocytes for each COVID-19 patient based on m6A levels and nine selected differentially expressed genes (m6A-DEGs) mostly related to the immune response; in this model, patients displaying higher [protective] scores showed a better prognosis related to T-cell activation compared to patients with lower scores; in addition to clinical prognosis, the model may predict the possibility of contracting COVID-19 in patients infected with SARS-CoV-2, as well as the detection of SARS-CoV-2 carriers [26]”.

• Li, N.; Hui, H.; Bray, B.; Gonzalez, G. M.; Zeller, M.; Anderson, K. G.; Knight, R.; Smith, D.; Wang, Y.; Carlin, A. F.; Rana, T.M. METTL3 regulates viral m6A RNA modification and host cell innate immune responses during SARS-CoV-2 infection. Cell Rep. 2021, 35, 109091. https://doi.org/10.1016/j.celrep.2021.109091
• An, S.; Xie, Z.; Liao, Y.; Jiang, J.; Dong, W.; Yin, F.; Li, W. X.; Ye, L.; Lin, J.; Liang, H. Systematic analysis of clinical relevance and molecular characterization of m6A in COVID-19 patients. Genes & diseases. 2022. 10.1016/j.gendis.2021.12.005. Advance online publication. https://doi.org/10.1016/j.gendis.2021.12.005
• Meng, Y.; Zhang, Q.; Wang, K.; Zhang, X.; Yang, R.; Bi, K.; Chen, W.; Diao, H. RBM15- mediated N6-methyladenosine Modification affects COVID-19 severity By Regulating The Expression Of Multitarget Genes. Cell Death Dis. 2021, 12 (8), 732. https://doi.org/10.1038/s41419-021-04012-z
• Qiu, X.; Hua, X.; Li, Q.; Zhou, Q.; Chen, J. m6A regulator-mediated methylation modification patterns and characteristics of immunity in blood leukocytes of COVID-19 patients. Front. Immunol. 2021, 12, 774776. https://doi.org/10.3389/fimmu.2021.774776

Reviewer 2 Report

This study investigates the levels of global m6A RNA methylation in people infected with distinct SARS-CoV-2 variants, the correlation of m6A with viral load or vaccination status. The authors used the m6A RNA Methylation Quantification Kit to determine the m6A levels and performed statistical analysis, made the conclusions that global m6A methylation differed across viral variants; most of COVID-19 patients had lower m6A levels than normal individuals; patients with complete vaccination schemes showed lower m6A levels compared to unvaccinated patients. The study of m6A modification of SARS-CoV-2 is a potentially interesting story. However, the conclusions are hard to explain and exist some weaknesses. First the sample size of the survey is too small. Secondly, it is hard to distinguish current m6A modifications from host RNA or virus RNA. From the conclusions, there are should be some host m6A modifications due to higher m6A levels of uninfected individuals by using nasopharyngeal samples. If there are some host m6A modifications, the current conclusions are not totally right. In addition, other related literature should be discussed.

Other points need to be addressed:

1. The current research status of SARS-CoV-2 m6A modification and effects of viral infection on host m6A modification should be introduced.
2. 3B: Scatter plot between m6A methylation levels is not relevant to the topic of this article.

Author Response

We want to thank the four reviewers for their comments and the time to revise this manuscript. All major changes in the document are highlighted in yellow, however several minor changes (not highlighted) were made throughout the document, and several references were added.

 Reviewer 2

1) The current research status of SARS-CoV-2 m6A modification and effects of viral infection on host m6A modification should be introduced.

Thank you for the suggestion. We searched the literature for SARS-CoV-2 m6A modifications and effects of viral infection on host m6A modifications, and added the following texts to the Introduction section:

“Following the discovery of RNA methylation at N6- position of adenosine (m6A), the most prevalent modification of RNA in mammalian cells [7,8], this modification has also been detected in several viral genomes [9]. Most m6A is found in the consensus sequence motif DRACH (where A* denotes the methylated adenosine, D denotes A, G or U, R denotes A and G, and H denotes A, C or U) and mRNAs may contain from one to up to 20 m6A sites or more [10,11].”

• Desrosiers, R.; Friderici, K.; Rottman, F. Identification of methylated nucleosides in messenger RNA from Novikoff hepatoma cells. Proc Natl Acad Sci U S A. 1974, 71, 3971–3975. https://doi.org/10.1073/pnas.71.10.3971
• Chen, M.; Wong, C.M. The emerging roles of N6-methyladenosine (m6A) deregulation in liver carcinogenesis. Mol Cancer. 2020, 19, 44. https://doi.or/10.1186/s12943-020-01172-y
• Manners, O.; Baquero-Perez, B.; Whitehouse, A. m6A: widespread regulatory control in virus replication. Biochim Biophys Acta Gene Regul Mech. 2019,1862, 370–381. https://doi.org/10.1016/j.bbagrm.2018.10.015
• Fu, Y.; Dominissini, Rechavi, G.; He, C. Gene expression regulation mediated through reversible m6A RNA methylation. Nat Rev Genet. 2014, 15, 293–306. https://doi.org/10.1038/nrg3724
• Ke, S.; Pandya-Jones, A.; Saito, Y.; Fak, J.J.; Vågbø, C.B.; Geula, S.; Hanna, J.H.; Black, D.L.; Darnell Jr, J.E.; Darnell, R.B. m6A mRNA modifications are deposited in nascent pre-mRNA and are not required for splicing but do specify cytoplasmic turnover. Genes Dev. 2017, 31, 990–1006. https://doi.org/10.1101/gad.301036.117

“…an increase in METTL3 expression 48 h post-infection in Vero E6 cells was positively associated with SARS-CoV-2 replication [15]. In accordance, depletion of METTL3 leads to a reduction in SARS-CoV-2 replication [13,15,16], although the opposite has also been reported [14]. Furthermore, knockdown of specific erasers (ALKBH5) and readers (YTHDF1, YTHDF2, YTHDF3) have also been shown to impact on viral replication [14,16].

Recent studies show that SARS-CoV-2 infection changes the host cell m6A methylome in vitro, promoting differential expression of host genes [13], and in vivo, altering m6A modification levels in lymphocytes from peripheral blood samples by increased expression of the m6A methyltransferase RNA-binding motif protein 15 (RBM15) [17], or decreased expression of METTL3 in epithelial cells of bronchoalveolar lavage fluid of COVID-19 patients [13].

To date, only a few studies have probed for alterations in global m6A levels in a limited number of COVID-19 positive individuals (n = 2 – 20) [13, 17, 18]. These studies found increased levels of m6A in peripheral blood samples [17] or in epithelial cells of bronchoalveolar lavage fluid [13] in infected individuals relative to non-infected individuals that are associated with increased expression of RBM15, that encodes for a protein that facilitates m6A by guiding METTL3 to the target RNA sequence [17].”

• Li, N.; Hui, H.; Bray, B.; Gonzalez, G. M.; Zeller, M.; Anderson, K. G.; Knight, R.; Smith, D.; Wang, Y.; Carlin, A. F.; Rana, T.M. METTL3 regulates viral m6A RNA modification and host cell innate immune responses during SARS-CoV-2 infection. Cell Rep. 2021, 35, 109091. https://doi.org/10.1016/j.celrep.2021.109091
• Liu, J.; Xu, Y.P.; Li, K.; Ye, Q.; Zhou, H.Y.; Sun, H.; Li, X.; Yu, L.; Deng, Y.Q.; Li, R.T.; Cheng, M.L.; He, B.; Zhou, J.; Li, X.F.; Wu, A.; Yi, C.; Qin, C.F. The m6A methylome of SARS-CoV-2 in host cells. Cell Res. 2021, 31, 404–414. https://doi.org/10.1038/s41422-020-00465-7
• Zhang, X.; Hao, H.; Ma, L.; Zhang, Y.; Hu, X.; Chen, Z.; Liu, D.; Yuan, J.; Hu, Z.; Guan, W. Methyltransferase-like 3 modulates severe acute respiratory syndrome coronavirus-2 RNA N6-methyladenosine modification and replication. mBio. 2021, 12, e01067-21. https://doi.org/10.1128/mBio.01067-21
• Burgess, H.M.; Depledge, D.P.; Thompson, L.; Srinivas, K.P.; Grande, R.C.; Vink, E.I.; Abebe, J.S.; Blackaby, W.P.; Hendrick, A.; Albertella, M.R.; Kouzarides, T.; Stapleford, K.A.; Wilson, A.C.; Mohr, I. Targeting the m6A RNA modification pathway blocks SARS-CoV-2 and HCoV-OC43 replication. Genes Dev. 2021, 35, 1005–1019. https://doi.org/10.1101/gad.348320.121
• Meng, Y.; Zhang, Q.; Wang, K.; Zhang, X.; Yang, R.; Bi, K.; Chen, W.; Diao, H. RBM15- mediated N6-methyladenosine Modification affects COVID-19 severity By Regulating The Expression Of Multitarget Genes. Cell Death Dis. 2021, 12 (8), 732. https://doi.org/10.1038/s41419-021-04012-z
• Qiu, X.; Hua, X.; Li, Q.; Zhou, Q.; Chen, J. m6 A Regulator-Mediated Methylation Modification Patterns and Characteristics of Immunity in Blood Leukocytes of COVID-19 Patients. Front. Immunol. 2021, 12, 774776. https://doi.org/3389/fimmu.2021.774776
• An, S.; Xie, Z.; Liao, Y.; Jiang, J.; Dong, W.; Yin, F.; Li, W. X.; Ye, L.; Lin, J.; Liang, H. Systematic analysis of clinical relevance and molecular characterization of m6A in COVID-19 patients. Genes & diseases. 2022. 10.1016/j.gendis.2021.12.005. Advance online publication. https://doi.org/10.1016/j.gendis.2021.12.005

2) 3B: Scatter plot between m6A methylation levels is not relevant to the topic of this article.

The reviewer is right. We eliminated the figure 3B, but we left the p value in the text to rule out the influence of age on m6A methylation.

Reviewer 3 Report

The manuscript postulates that m6A RNA methylation may be an epigenetic mechanisms used by the SARS CoViD virus to modulate infection. Though a lot of follow up studies are required, the manuscript lays a good groundwork understanding the mechanism in which the virus works. I have a few comments:

1. The sample for negative control was only 10. The statistics could be better if you used more number for the control samples.

2. It will be interesting to include a diagram to show the divergence between the variants to appreciate the differential methylation pattern.

Author Response

We want to thank the four reviewers for their comments and the time to revise this manuscript. All major changes in the document are highlighted in yellow, however several minor changes (not highlighted) were made throughout the document, and several references were added.

 Reviewer 3

1) The sample for negative control was only 10. The statistics could be better if you used more number for the control samples.

We agree with the reviewer. Since the initial experimental design was balanced, we only considered 10 samples per treatment (10 for each viral variant and 10 for the negative control), because of the availability of positive samples of each variant, to make a balanced design. With this sample size, significant differences were observed. We recommend increasing the sample size per treatment for future studies and this consideration was included in the manuscript, at the end of the discussion: “…increasing the sample size of analyzed individuals is important in order to improve the reliability of the conclusions presented”.

2) It will be interesting to include a diagram to show the divergence between the variants to appreciate the differential methylation pattern.

Figure 2A of the manuscript shows the differential methylation patterns by viral variant. In addition, we included a graphical abstract showing this behaviour in a more schematic manner. The reviewer is right and the schematic representation of m6A methylation in different variants should be included. Therefore we decided to include the graphical abstract also in the main manuscript (as figure 5) to illustrate this behaviour.

Reviewer 4 Report

This is an interesting manuscript regarding the association between the SARS-CoV-2 infection and RNA m6A methylation. Through analysis of nasopharyngeal sample, authors revealed that global m6A methylation differed significantly across viral variants, the delta and omicron being extremely low. Furthermore, individuals with complete vaccination showed lower m6A methylation levels than unvaccinated individuals. Collectively, these preliminary results provide original and solid evidence that alterations in global m6A methylation are closely related with SARS-CoV-2 infection. While, some minor revisions are needed before it could be accepted for publication.

Comments:

1.       Since authors used the nucleocapsid gene expression for test, more specific functions of the N protein should be discussed in the introduction part.

2.       Rapid changes of the dominant strain over the sampling period deserves a paragraph in discussion.

3.       In the discussion part, it would be better to provide even deeper discussions for the discovered phenomena. For example, whether the m6A level is connected to the severity of COVID-19 patients? Whether the low m6A level in omicron contribute to its strong ability to escape host immune defense?

4.       Line numbers in table1, figure2 and figure3 are abnormal. Please check the format carefully.

5.       For some sentences, appropriate citations are missing. E.g., Line 51, needs a reference; Line 60 needs refs.

Author Response

We want to thank the four reviewers for their comments and the time to revise this manuscript. All major changes in the document are highlighted in yellow, however several minor changes (not highlighted) were made throughout the document, and several references were added.

 Reviewer 4

1) Since authors used the nucleocapsid gene expression for test, more specific functions of the N protein should be discussed in the introduction part.

Thank you for the suggestion. We included the following text in the Introduction section:

“The N protein is a crucial structural component of SARS-CoV-2 that participates in the virion assembly and is often used as a diagnostic marker of viral infection [2]. In a vaccine setting, the N protein induces SARS-CoV-2-specific T cell proliferation and cytotoxic activity and promotes long-lasting T cell immunity [3].”

• Bai, Z.; Cao, Y.; Liu, W.; Li, J. The SARS-CoV-2 Nucleocapsid Protein and Its Role in Viral Structure, Biological Functions, and a Potential Target for Drug or Vaccine Mitigation. Viruses. 2021, 13 (6), 1115. https://doi.org/10.3390/v13061115
• Le Bert, N.; Tan, A.T.; Kunasegaran, K.; Tham, C.Y.L.; Hafezi, M.; Chia, A.; Chng, M.H.Y.; Lin, M.; Tan, N.; Linster, M.; Chia, W. N.; Chen, M. I.; Wang, L. F.; Ooi, E. E.; Kalimuddin, S.; Tambyah, P. A.; Low, J. G.; Tan, Y. J.; Bertoletti, A. SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls. Nature. 2020, 584, 457–462. https://doi.org/10.1038/s41586-020-2550-z

2) Rapid changes of the dominant strain over the sampling period deserves a paragraph in discussion.

Thank you for the observation. The port of Mazatlán is a touristic location, receiving every year thousands of visitors from Mexico, South America, USA, Canada, and other countries around the world. Rapid changes of the dominant variants over the sampling period could be due to the crowded environment, allowing the virus to spread. In addition, high mutation and recombination rates of RNA viruses, and asymptomatic infections could also contribute to the rapid diversification of SARS-CoV-2 (Duarte et al., 2022), even among fully vaccinated individuals. This paragraph was included in the Discussion section.

• Duarte, C.M.; Ketcheson, D.I.; Eguíluz, V.M.; Agustí, S.; Fernández-Gracia, J.; Jamil, T.; Laiolo, E.; Gojobori, T.; Alam, I. Rapid evolution of SARS-CoV-2 challenges human defenses. Sci Rep. 2022, 12, 6457. https://doi.org/10.1038/s41598-022-10097-z

3) In the discussion part, it would be better to provide even deeper discussions for the discovered phenomena. For example, whether the m6A level is connected to the severity of COVID-19 patients? Whether the low m6A level in omicron contribute to its strong ability to escape host immune defense?

Thank you for these suggestions. We searched the literature and included the following information in the Discussion:

“To our knowledge, this is the first report showing that global m6A levels of nasopharyngeal RNA samples of patients infected with SARS-CoV-2 differ among viral variants. Interestingly, the two most contagious variants (delta and omicron) - with omicron being the most contagious globally- [22] showed the lowest methylation levels. These differences are unlikely to reflect variation in disease severity across variants as COVID-19 patients with beta and delta variants generally are at higher risk of developing severe disease compared to patients with alpha, gamma [23], and omicron variant(s), whose mutations have been suggested to contribute to the host immune escape [24]. Interestingly, DRACH motifs are highly conserved among SARS-CoV-2 variants [25], thus the contribution of DRACH motifs in differential methylation levels among variants remains unclear and require further research.

From a public health perspective, identifying a reliable marker of severe COVID-19 risk is of utmost importance. Unfortunately, we have no information regarding disease severity of the individuals analyzed in this study since most samples were from outpatients. In addition, we found no correlation between viral load and m6A levels across variants. However, previous studies have reported a higher m6A level in lung or peripheral blood from patients with moderate or severe COVID-19 disease compared to healthy individuals [13, 17, 18, 26]. Interestingly, in peripheral blood, these changes were associated with increased expression of RBM15 [17]. Moreover, Qiu et al. [26] proposed a predictive “m6A score” to quantify and model the m6A pattern in blood leukocytes for each COVID-19 patient based on m6A levels and nine selected differentially expressed genes (m6A-DEGs) mostly related to the immune response; in this model, patients displaying higher [protective] scores showed a better prognosis related to T-cell activation compared to patients with lower scores; in addition to clinical prognosis, the model may predict the possibility of contracting COVID-19 in patients infected with SARS-CoV-2, as well as the detection of SARS-CoV-2 carriers [26].“

• da Silva, S.; de Lima, S. C.; da Silva, R. C.; Kohl, A.; Pena, L. Viral load in COVID-19 patients: implications for prognosis and vaccine efficacy in the context of emerging SARS-CoV-2 variants. Front Med (Lausanne). 2022, 8, 836826. https://doi.org/10.3389/fmed.2021.836826
• McCallum, M.; Czudnochowski, N.; Rosen, L.E.; Zepeda, S.K.; Bowen, J.E.; Walls, A.C.; Hauser, K.; Joshi, A.; Stewart, C.; Dillen, J.R; Powell, A.E.; Croll, T.I.; Nix, J.; Virgin, H.W.; Corti, D.; Snell, G.; Veesler, D. Structural basis of SARS-CoV-2 Omicron immune evasion and receptor engagement. Science. 2022, 375, 864–868. https://doi.org/1126/science.abn865
• Campos, J.H.C.; Maricato, J.T.; Braconi, C.T.; Antoneli, F.; Janini, L.M.R.; Briones, M.R.S. Direct RNA sequencing reveals SARS-CoV-2 m6A sites and possible differential drach motif methylation among variants. Viruses. 2021, 13, 2108. https://doi.org/10.3390/v13112108
• Qiu, X.; Hua, X.; Li, Q.; Zhou, Q.; Chen, J. m6 A Regulator-Mediated Methylation Modification Patterns and Characteristics of Immunity in Blood Leukocytes of COVID-19 Patients. Front. Immunol. 2021, 12, 774776. https://doi.org/3389/fimmu.2021.774776
• McCallum, M.; Czudnochowski, N.; Rosen, L.E.; Zepeda, S.K.; Bowen, J.E.; Walls, A.C.; Hauser, K.; Joshi, A.; Stewart, C.; Dillen, J.R; Powell, A.E.; Croll, T.I.; Nix, J.; Virgin, H.W.; Corti, D.; Snell, G.; Veesler, D. Structural basis of SARS-CoV-2 Omicron immune evasion and receptor engagement. Science. 2022, 375, 864–868. https://doi.org/1126/science.abn865
• Li, N.; Hui, H.; Bray, B.; Gonzalez, G. M.; Zeller, M.; Anderson, K. G.; Knight, R.; Smith, D.; Wang, Y.; Carlin, A. F.; Rana, T.M. METTL3 regulates viral m6A RNA modification and host cell innate immune responses during SARS-CoV-2 infection. Cell Rep. 2021, 35, 109091. https://doi.org/10.1016/j.celrep.2021.109091
• Meng, Y.; Zhang, Q.; Wang, K.; Zhang, X.; Yang, R.; Bi, K.; Chen, W.; Diao, H. RBM15- mediated N6-methyladenosine Modification affects COVID-19 severity By Regulating The Expression Of Multitarget Genes. Cell Death Dis. 2021, 12 (8), 732. https://doi.org/10.1038/s41419-021-04012-z
• An, S.; Xie, Z.; Liao, Y.; Jiang, J.; Dong, W.; Yin, F.; Li, W. X.; Ye, L.; Lin, J.; Liang, H. Systematic analysis of clinical relevance and molecular characterization of m6A in COVID-19 patients. Genes & diseases. 2022. 10.1016/j.gendis.2021.12.005. Advance online publication. https://doi.org/10.1016/j.gendis.2021.12.005

4) Line numbers in table1, figure2 and figure3 are abnormal. Please check the format carefully.

The revised version of the manuscript (which we received from the journal office in word format) does not include line numbers. Line numbers are generated in the pdf version of the document and we are unable to edit or re-format.

5) For some sentences, appropriate citations are missing. E.g., Line 51, needs a reference; Line 60 needs refs.

The reviewer is right. We added the references:

• Davies, N.G.; Abbott, S.; Barnard, R.C.; Jarvis, C.I.; Kucharski, A.J.; Munday, J.D.; Pearson, C.A.B.; Russell, T.W.; Tully, D.C.; Washburne, A.D.; Wenseleers, T.; Gimma, A.; Waites, W.; Wong, K.L.M.; van Zandvoort, K.; Silverman, J.D.; CMMID COVID-19 Working Group; COVID-19 Genomics UK (COG-UK) Consortium; Diaz-Ordaz, K.; Keogh, R.; Eggo, R.M.; Funk, S.; Jit, M.; Atkins, K.E.; Edmunds, W.J. Estimated transmissibility and impact of SARS-CoV-2 lineage B.1.1.7 in England. Science. 2021,372, eabg3055. https://doi.org/10.1126/science.abg3055
• Bager, P.; Wohlfahrt, J.; Fonager, J.; Rasmussen, M.; Albertsen, M.; Michaelsen, T. Y.; Møller, C. H.; Ethelberg, S.; Legarth, R.; Button, M.; Gubbels, S.; Voldstedlund, M.; Mølbak, K.; Skov, R. L.; Fomsgaard, A.; Krause, T. G.; Danish Covid-19 Genome Consortium. Risk of hospitalisation associated with infection with SARS-CoV-2 lineage B.1.1.7 in Denmark: an observational cohort study. Lancet Infect Dis. 2021, 21(11), 1507–1517. https://doi.org/10.1016/S1473-3099(21)00290-5
• Le Bert, N.; Tan, A.T.; Kunasegaran, K.; Tham, C.Y.L.; Hafezi, M.; Chia, A.; Chng, M.H.Y.; Lin, M.; Tan, N.; Linster, M.; Chia, W. N.; Chen, M. I.; Wang, L. F.; Ooi, E. E.; Kalimuddin, S.; Tambyah, P. A.; Low, J. G.; Tan, Y. J.; Bertoletti, A. SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls. Nature. 2020, 584, 457–462. https://doi.org/10.1038/s41586-020-2550-z

Round 2

Reviewer 2 Report

None.