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  1. Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

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    Reply to the reviewers

    We thank the both the reviewers for their constructive comments. Please see our point-by-point response to all the comments.

    *Reviewer #1 (Evidence, reproducibility and clarity (Required)): *

    • Summary *
    • The authors of this manuscript confirm data found by others by determining replication kinetics of the ancestral B.6 SARS-CoV-2 virus, Delta and Omicron BA.1 and BA.2 in Calu-3 cells. The authors quantify barrier integrity between variants and interferon induction to conclude that Delta is more cytopathic and induced less interferon than Omicron, possibly leading to its increased pathogenesis. In addition the authors identify CuCl2 and FeSO4 as potential antivirals. *

    *Major comments *

    1. *__Reviewer comment: __The author's argue that Omicron's slower replication on Calu-3 cells correlates with mild disease, however many publications show that Omicron replicates more efficiently/ rapidly in primary human airway cultures: *

    *__Reviewer comment: __Can the authors explain why air-liquid grown Calu-3 cells appear to display similar viral titers for Omicron and Delta at 24 and 36 h.p.i (Figure 5B), however lower viral replication in Figure 3B? If the cells in Figure 3B are submerged, then the authors should identify why ALI grown Calu-3 cells are more susceptible to Omicron. *

    Response: Cells were grown in plastic multi-well plates for growth curve experiments shown in Figure 3. The cells in this condition are not polarized and the virus titers are the total amount of virus released into the culture supernatant. The infection conditions in Figure 5 is under air-liquid culture conditions, from polarized cells. Therefore, the virus titers are only from the basolateral chamber. The outcomes of figure 3 and figure 5 are not comparable due to these technical differences. We will add this explanation in the results section.

    *__Reviewer comment: __The authors suggest that Delta disrupts epithelial barrier integrity to a larger extent compared to B.6 and Omicron, however this may be due to fewer infected cells (despite equal viral titers, the nucleocapsid staining in Figure 2 and 5C suggests fewer infected cells). Have the authors imaged B.6 or Omicron at a later timepoint (or normalized virus input for equal infected cells) to determine barrier integrity when the amount of infected cells is equal? Alternatively, the authors should discuss this as a possible limitation of their study, especially since they argue this is a major reason why Delta has a growth advantage (lines 345 to 349). *

    Response: We performed confocal imaging of transwells from air-liquid interface model using a 20X objective and have obtained data to show that the percent of infected cells is similar between Omicron and Delta variant. We will include this data in the revised manuscript. In an in vitro system, once the infection is set in, the infected cells eventually die and the TEER reaches background levels. We are proposing a delay in disruption of barrier integrity most probably due to lower cytopathogenicity of the Omicron variant. As per the reviewer’s suggestion, we will discuss the possible limitation of the models and provide additional interpretations.

    Minor comments *A) __Reviewer comment: __Line 118: Implications of this sentence are too strong. The authors have not shown the causality of Ct values and transmission, therefore they should reword the sentence: "indicating a high viral burden in patients during this period resulting in increased transmission of the virus among the contacts" to "likely attributing to increased transmission..." *

    Response: We will correct this.

    *__B) Reviewer comment: __Line 289: The authors suggest that infection with the Omicron variant generated higher levels of antibodies to the Delta variant, however these individuals are already vaccinated and elicit cross-neutralizing antibodies against Delta even before their Omicron infection. Therefore the Delta response is boosted and the Omicron response is essentially a primary response since vaccination elicits almost no cross-protection in itself. Therefore the authors should compare primary Delta infected individuals to primary Omicron infected individuals to determine cross-protection levels. *

    Response: We agree with the reviewer’s argument. Please note that the two vaccines used in India are against the ancestral virus (inactivated) or the spike protein expressed by the adenovirus vector backbone. As over 90% of the population in India have been fully vaccinated with these two vaccines and a majority of them may also have been infected with delta variant and now with omicron, it is practically impossible to compare primary delta cases vs primary omicron cases at this stage. As part of another study in mid 2021, after the second wave of COVID-19 infections due to the Delta variant in India, we randomly selected 55 samples which had a detectable FRNT50 value for the delta variant, to test for their ability to neutralize the Omicron variant. Only twenty of the 55 samples had detectable levels of neutralizing antibodies against the Omicron variant. By assigning a FRNT50 value of 10 for the samples which had no detectable levels of antibodies in the starting dilution (1:20) of the assay, we obtained a GMT of 22.5 (95% CI: 16, 31) for these 55 samples. This value was 20-fold lower than the GMT of Delta variant which was 404 (95% CI:248, 658). This clearly indicates that even during the peak of delta wave, there were barely any cross-reactive antibodies to the Omicron variant. This study was recently published [NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-31170-1]. It would be interesting to eventually compare the antibody responses in reinfections with other sub-lineages of Omicron variant which is beyond the scope of our manuscript. We will add this description in the results and discussion section of the revised manuscript.

    *C) __Reviewer comment: __There appears to be no reference to Figure 6G, however this reference is most likely missing from line 306. *

    Response: Thank you for bringing this to our notice. We will insert the reference to Figure 6G.

    *D) __Reviewer comment: __Line 359-362: The authors suggest that waning antibody titers increase susceptibility to new variants of concern, however their cohort already possessed very low antibody titers against Omicron a month after vaccination (Figure 7F) suggesting they could be equally susceptible to Omicron 1 and 6 months after vaccination. *

    Response: Please note that nine out of 15 samples had FRNT50 value above the level of detection after vaccination in June 2021. The number of samples positive for Omicron antibodies reduced to six out of 15 by Dec 2021 suggesting that relatively more people were without protective antibodies for Omicron variant by Dec 2021. Around 70% of the population was seropositive by Aug 2021 (https://doi.org/10.1016/j.ijid.2021.12.353) and most adults in India received both doses of their vaccine after June 2021 which would have boosted the humoral and cellular response to SARS-CoV-2. This is corroborated in a recently published report, where we showed that 36 out of 55 previously infected subjects had neutralizing antibodies for the Omicron variant after receiving a single dose of inactivated vaccine. Therefore, in the context of hybrid immunity in India, we speculate that waning antibody titers could have played a significant role in the emergence and spread of Omicron variant in addition to the ability of the Omicron variant to escape neutralization, replicate more efficiently in the upper respiratory tract etc., The fact that booster doses of vaccines developed against the ancestral virus/viral protein was capable of increasing the level of neutralizing antibodies to omicron variant suggests that the level of antibodies above a certain threshold may play a significant role in protecting against the omicron variant.

    Reviewer #1 (Significance (Required)):

    • __Reviewer comment: __Many of the conclusions based on replication and barrier integrity may not represent the situation in primary human tissues and does not explain the rapid spread of Omicron. In addition, interferon induction has already been described for these variants and this finding is not novel. The manuscripts most interesting and novel finding is the role of CuCl2 and FeSO4 as antivirals. It would be interesting to test these salts in primary human airway cultures. *

    Response: The study was conducted in the months of Jan-March 2022 and the first version of the results were uploaded on a preprint server in March 2022. The process of journals handling the manuscript and obtaining reviews is not under our control. We cannot argue to defend the comments on novelty when the Omicron variant is barely six months old and new variants continue to emerge. The deluge of publications should not result in reviewers branding most of the efforts as not novel or insignificant. We have been trying since three months to obtain primary cells but the distributors are unable to supply the same. We will continue to try to obtain cells from one or the other source. Transwells are back-ordered with expected delivery dates in three months. Meanwhile, we now have HBEC3-KT cells which are normal human bronchial epithelial cells immortalized with CDK4 and hTERT. We will perform the inhibition experiments in these cell lines to convince the reviewers.

    __Reviewer #2 (Evidence, reproducibility and clarity (Required)): __

    *In the manuscript entitled "BA.1 and BA.2 sub-lineages of Omicron variant have comparable replication kinetics and susceptibility to neutralization by antibodies" the authors assess the kinetics of growth of SARS-CoV-2 variants in Calu-3 cells and their effects on epithelial junction, and the interferon response. The authors also analyze the capacity of metal salts to block SARS CoV-2 replication in Calu-3 cells. Finally, the authors characterize the ability of vaccinated and/or COVID-19 patients to develop neutralizing antibodies to different variants using FRNT and specific binding assays (ELISA). *

    • The paper largely confirms several previous reports on the replication capacity and interferon responses of the different variants. Although the title and abstract focus on the Omicron sub-lineages, the paper is mostly focused on comparing original CoV2, with Kappa, Delta and Omicron. *

    • Figures 1-5 compare the replication kinetics, interferon responses, and epithelial barrier disruption of Kappa, Delta and the original Omicron (B.1.1.529) to the original B6 variant. On a separate note, Figure 7 shows the ability of metal salts (especially iron, copper, and zinc) to block viral RNA-dependent RNA polymerase activity (RdRp) in vitro. The authors also show the effect on virus replication in Calu-3 cells (Delta and Omicron B.1.1.529 only). The data mainly focus on the variants, the Delta and the Omicron (BA.1.1.529 and not the BA.1 and BA.2 sub-lineages) except in Fig 6A, B, G. *

    • __Reviewer comment: __Most importantly, a major limitation of the paper is that when human samples are analyzed, the authors assume that the patients have been infected with a specific variant according to the "peak" of infection, but sequencing is never performed. When neutralization and binding of antibodies are analyzed, the information on the patients is unclear - for example, were the patients exposed to Delta or Omicron or any of their sub-lineages? What was the vaccination status of SARS CoV-2 positive patients? And why non-tested individuals showing symptoms were included in the study (lines 302-304)? *

    Response: We thank the reviewer for the comments. Over 90% of the population in India is vaccinated. All the participants of the study have been vaccinated in 2021. The participants were enrolled into the study almost 4 weeks after recovery from illness. We have enrolled participants who have reported to have had fever or COVID-19-like symptoms in the preceding weeks with or without confirmed RT-PCR test results. Testing is an individual and voluntary choice now. Therefore, it would be difficult to find RT-PCR confirmed cases. Our assumption about exposure is based on a nationwide sequencing effort of thousands of samples every week and this approach is reliable and credible. As indicated in the text and in the supplementary figure, Omicron lineages BA.1 followed by BA.2 were the circulating virus lineages since Jan 2021 in India.

    *__Reviewer comment: __The authors show that BA.1 and BA.2 have similar replication kinetics in Calu-3 cells and induce similar neutralizing antibodies in the patients tested. However, there is a large disconnection with the rest of the paper that is mostly focused on Kappa, Delta, and Omicron B.1.1.529. Also, no comparisons between these variants and BA.1 or BA.2 have been shown. Similarly, a large assumption in the paper is that the patients who tested positive for COVID-19 have had "natural Omicron infection" (lines 36-37; lines 307-311) when it could be any other variants or Omicron sub-lineages as well. *

    Response: Please note that the B.1.1.529 which was used at the beginning of the study is the BA.1 sub-lineage which has been compared with Kappa and Delta variants. BA.2 emerged at later stages and therefore we have compared the kinetics and neutralization titer between BA.1 and BA.2. It is unreasonable to expect to repeat all the comparisons with BA.2 considering the cost and challenges of working in a BSL-3 environment. The initial version of this data was uploaded on preprint server in March 2022 when only two sub-lineages of Omicron namely BA.1 and BA.2 existed. Our data from the national SARS-CoV-2 sequencing consortium clearly shows that there were no other sub-lineages circulating at that time.

    Reviewer #2 (Significance (Required)):

    *__Reviewer comment: __In light of the fact that most of the paper does not look at the subvariants BA.1 and BA.2 of Omicron- either the authors compare BA.1 and BA.2 more comprehensively with Omicron B.1.1.529 or rewrite the conclusions and claims of the current paper. Similar to the experiments comparing B6 with Kappa, Delta and Omicron, Omicron B.1.1.529 should be compared similarly to BA.1 and BA.2 in a separate figure. In any case, the novelty compared to other papers -also cited by the authors- remains limited. *

    Response: We will revise the conclusions and claims of the paper as per the suggestions. Please see our response to reviewer 1 with regards to the novelty of our observations. The B.1.1.529 variant was later classified as the BA.1 variant. Our study was uploaded on the preprint server in March 2022 and the entire review process has taken four months. It is unfair to now demand comparison of BA.2 with Kappa or Delta variant which does not add any additional value to our observations.

    *__Reviewer comment: __In addition to the concerns mentioned above, there are more pressing variants circulating right now, such as BA.4 and BA.5. These variants are not referred in the paper. It might be beyond the scope of the paper, but including more analyses with BA.1, BA.2 (as the ones done with B.1.1.529) and adding some key data with BA.3, BA.4, BA.5 might substantially increase the relevance and importance of the paper. *

    Response: Please see our comments above. Our efforts are continuing in this direction to further look at antibody responses and replication kinetics of newer variants which have emerged recently. However, the scarcity of positive clinical samples and lower probability of getting samples that would be suitable for virus isolation are the challenges we are dealing with. We think testing newer variants which have emerged during the review process is certainly valuable but is extremely difficult under the current circumstances. We will have to apply to seek import permits to obtain these sub-lineages or enrol patients with symptoms and keep testing them to isolate, culture the virus and obtain whole genome sequence. We will have to establish neutralization assays with newer sub-variants to test in parallel with other Omicron lineages. All this is beyond the scope of our manuscript and will take few months of paper work and experimentation.

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  2. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #2

    Evidence, reproducibility and clarity

    In the manuscript entitled "BA.1 and BA.2 sub-lineages of Omicron variant have comparable replication kinetics and susceptibility to neutralization by antibodies" the authors assess the kinetics of growth of SARS-CoV-2 variants in Calu-3 cells and their effects on epithelial junction, and the interferon response. The authors also analyze the capacity of metal salts to block SARS CoV-2 replication in Calu-3 cells. Finally, the authors characterize the ability of vaccinated and/or COVID-19 patients to develop neutralizing antibodies to different variants using FRNT and specific binding assays (ELISA).

    The paper largely confirms several previous reports on the replication capacity and interferon responses of the different variants. Although the title and abstract focus on the Omicron sub-lineages, the paper is mostly focused on comparing original CoV2, with Kappa, Delta and Omicron. Figures 1-5 compare the replication kinetics, interferon responses, and epithelial barrier disruption of Kappa, Delta and the original Omicron (B.1.1.529) to the original B6 variant. On a separate note, Figure 7 shows the ability of metal salts (especially iron, copper, and zinc) to block viral RNA-dependent RNA polymerase activity (RdRp) in vitro. The authors also show the effect on virus replication in Calu-3 cells (Delta and Omicron B.1.1.529 only). The data mainly focus on the variants, the Delta and the Omicron (BA.1.1.529 and not the BA.1 and BA.2 sub-lineages) except in Fig 6A, B, G.

    Most importantly, a major limitation of the paper is that when human samples are analyzed, the authors assume that the patients have been infected with a specific variant according to the "peak" of infection, but sequencing is never performed. When neutralization and binding of antibodies are analyzed, the information on the patients is unclear - for example, were the patients exposed to Delta or Omicron or any of their sub-lineages? What was the vaccination status of SARS CoV-2 positive patients? And why non-tested individuals showing symptoms were included in the study (lines 302-304)?

    The authors show that BA.1 and BA.2 have similar replication kinetics in Calu-3 cells and induce similar neutralizing antibodies in the patients tested. However, there is a large disconnection with the rest of the paper that is mostly focused on Kappa, Delta, and Omicron B.1.1.529. Also, no comparisons between these variants and BA.1 or BA.2 have been shown. Similarly, a large assumption in the paper is that the patients who tested positive for COVID-19 have had "natural Omicron infection" (lines 36-37; lines 307-311) when it could be any other variants or Omicron sub-lineages as well.

    Significance

    In light of the fact that most of the paper does not look at the subvariants BA.1 and BA.2 of Omicron- either the authors compare BA.1 and BA.2 more comprehensively with Omicron B.1.1.529 or rewrite the conclusions and claims of the current paper. Similar to the experiments comparing B6 with Kappa, Delta and Omicron, Omicron B.1.1.529 should be compared similarly to BA.1 and BA.2 in a separate figure. In any case, the novelty compared to other papers -also cited by the authors- remains limited.

    In addition to the concerns mentioned above, there are more pressing variants circulating right now, such as BA.4 and BA.5. These variants are not referred in the paper. It might be beyond the scope of the paper, but including more analyses with BA.1, BA.2 (as the ones done with B.1.1.529) and adding some key data with BA.3, BA.4, BA.5 might substantially increase the relevance and importance of the paper.

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  3. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons


    Referee #1

    Evidence, reproducibility and clarity

    Summary

    The authors of this manuscript confirm data found by others by determining replication kinetics of the ancestral B.6 SARS-CoV-2 virus, Delta and Omicron BA.1 and BA.2 in Calu-3 cells. The authors quantify barrier integrity between variants and interferon induction to conclude that Delta is more cytopathic and induced less interferon than Omicron, possibly leading to its increased pathogenesis. In addition the authors identify CuCl2 and FeSO4 as potential antivirals.

    Major comments

    1. The author's argue that Omicron's slower replication on Calu-3 cells correlates with mild disease, however many publications show that Omicron replicates more efficiently/ rapidly in primary human airway cultures: Hui et al., (Nature, 2022) doi: https://doi.org/10.1038/s41586-022-04479-6 Peacock et al., (bioRxiv) doi: https://doi.org/10.1101/2021.12.31.474653 Lamers et al., (bioRxiv) doi: https://doi.org/10.1101/2022.01.19.476898
    2. Can the authors explain why air-liquid grown Calu-3 cells appear to display similar viral titers for Omicron and Delta at 24 and 36 h.p.i (Figure 5B), however lower viral replication in Figure 3B? If the cells in Figure 3B are submerged, then the authors should identify why ALI grown Calu-3 cells are more susceptible to Omicron.
    3. The authors suggest that Delta disrupts epithelial barrier integrity to a larger extent compared to B.6 and Omicron, however this may be due to fewer infected cells (despite equal viral titers, the nucleocapsid staining in Figure 2 and 5C suggests fewer infected cells). Have the authors imaged B.6 or Omicron at a later timepoint (or normalized virus input for equal infected cells) to determine barrier integrity when the amount of infected cells is equal? Alternatively, the authors should discuss this as a possible limitation of their study, especially since they argue this is a major reason why Delta has a growth advantage (lines 345 to 349).

    Minor comments

    Line 118: Implications of this sentence are too strong. The authors have not shown the causality of Ct values and transmission, therefore they should reword the sentence: "indicating a high viral burden in patients during this period resulting in increased transmission of the virus among the contacts" to "likely attributing to increased transmission..."

    Line 289: The authors suggest that infection with the Omicron variant generated higher levels of antibodies to the Delta variant, however these individuals are already vaccinated and elicit cross-neutralizing antibodies against Delta even before their Omicron infection. Therefore the Delta response is boosted and the Omicron response is essentially a primary response since vaccination elicits almost no cross-protection in itself. Therefore the authors should compare primary Delta infected individuals to primary Omicron infected individuals to determine cross-protection levels. There appears to be no reference to Figure 6G, however this reference is most likely missing from line 306.

    Line 359-362: The authors suggest that waning antibody titers increase susceptibility to new variants of concern, however their cohort already possessed very low antibody titers against Omicron a month after vaccination (Figure 7F) suggesting they could be equally susceptible to Omicron 1 and 6 months after vaccination.

    Significance

    Many of the conclusions based on replication and barrier integrity may not represent the situation in primary human tissues and does not explain the rapid spread of Omicron. In addition, interferon induction has already been described for these variants and this finding is not novel. The manuscripts most interesting and novel finding is the role of CuCl2 and FeSO4 as antivirals. It would be interesting to test these salts in primary human airway cultures.

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  4. SciScore for 10.1101/2022.01.28.22269990: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    EthicsIRB: Human Ethics: The study was approved by the Institutional ethics committees for human research at ESIC Hospital and Medical College (No.134/R/10/IEC/22/2021/02) and THSTI (THS 1.8.1/ (93)).
    Consent: Informed consent was obtained from all the participants.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Total RNA was isolated to detect SARS-CoV-2 using COVIDsure multiplex real time RT-PCR kit (Trivitron Healthcare) either at Employees State Insurance Corporation (ESIC) Medical College & Hospital or at the bioassay laboratory Translational Health Science and Technology Institute, Faridabad.
    Trivitron Healthcare
    suggested: None
    Statistical analysis: Data was analysed and final graphs were prepared using GraphPad Prism (Version 9) software.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).


    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.


    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.


    Results from JetFighter: We did not find any issues relating to colormaps.


    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.


    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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