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    Reviewer response

    We thank the reviews for the careful reviews, and were delighted to see that they assessed both the quality and significance of the work so highlty.

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

    *The authors investigated the cross-neutralization capacity of serum antibodies to past and future 229E coronaviruses using 229E spikes isolated from five time points and sera from two different periods (1985-1995 and 2020). They demonstrated a general pattern of asymmetric cross-neutralization, with sera cross-reactive to historical but not future strains. Using chimeras, the authors showed this pattern was mostly driven by antibodies to the evolving RBD. The rate of change in the neutralization titer, a possible measure of antigenic evolution, was estimated to be on par with that of flu B viruses. Interesting differences in individuals' cross-neutralization capacity were observed. The main take-away is that reinfection with 229E is enabled by antigenic escape, not "weak" immunity after infection (as proposed by others). *

    Thanks for the excellent summary of the paper. We agree with it, although we would note that our work does not exclude “weak immunity” as a possible compounding explanation for re-infection in addition to the antigenic evolution we demonstrate.

    **Major comments:**

    The key conclusions are convincing and justified by the data. The work is clearly presented and presented with sufficient detail for reproducibility. Characteristically and laudably, the authors have made all the code and data publicly available on GitHub.

    Thanks for the favorable summary.

    **Minor comments:**

    p 3: Perhaps it is clearer to write that 229E has been identified/isolated in humans for >50 y? Or do you really mean to imply (by contrast with "circulated") that NL63 emerged very recently?

    This is a good suggestion. We really do not know how long either CoV-229E or CoV-NL63 have been circulating humans, only that CoV-229E was first isolated >50 years ago whereas CoV-NL63 was first identified only in 2003. It is possible both viruses have been circulating for longer than that. We have made the suggested change to clarify.

    p 3: An important citation for the antigenic implications of the ladder-like phylogeny AND phylogenetic clustering by date is the classic paper introducing phylodynamics by Grenfell et al. (2004, Science).

    Thanks for pointing out this citation; we have added it.

    p 4: I might not be like all readers, but I prefer to see a bit in the main text about the source of sera for this kind of study. (I wonder about age, if donors are healthy, etc.)

    This is a good question, and we have expanded on it in both the main text and the methods. Briefly, the sera were all from apparently healthy individuals, and no information about recent respiratory virus infections were available. We have provided the age of the serum donor (at the time of serum collection) above the title of each plot showing person-specific neutralization data.

    p 4: "Our reason for focusing..." stops short. Is the idea that these are probably people who were recently infected?

    This is a good question, and we have elaborated in the revised text. We don’t have any direct information on whether the individuals had recent infections, although that seems plausible. More pragmatically, we reasoned that sera that had reasonably high initial titers would provide better dynamic range to see how titers changed as the virus evolved given our assay has a lower limit of detection.

    p 5: Probably my biggest suggestion for the paper is that it mention another relevant study. In 1980, Anne Underwood demonstrated similar asymmetric cross-immunity among early strains of H3N2 (but using rabbits, not human sera), finding that antibodies raised to one strain reacted more strongly by HAI to past strains than to later strains (doi: 10.1128/IAI.27.2.397-404.1980). This relates to the significance of the paper (next section).

    Thanks, this is a good and relevant citation, and we have added it when we discuss the possible asymmetry of antigenic change with respect to time.

    Obviously, there are citations to update throughout due to the booming SARS-CoV-2 literature.

    We have updated the other citations to keep pace with the fast-changing literature!

    Reviewer #1 (Significance (Required)):

    This study, if anything, undersells itself. Obviously it is a huge contribution to our understanding of how a seasonal coronavirus that bears important phenotypic resemblance to SARS-CoV-2 evolves, but I think it is also providing a foundational piece of evidence--a mechanism--of how rapid viral turnover (by antigenic evolution) occurs. There is no reason to think this should be limited to the coronaviruses, and I suspect the evidence here will go a long way to unifying the evolutionary and epidemiological dynamics of fast-evolving viruses.

    Thanks for the praise of the manuscript. Indeed, we were surprised to find that no similarly designed studies have been done even for influenza virus, and so are now interested in expanding our future work to do that as we fully agree it could provide insight more broadly.

    Asymmetric competition is nearly an ecological requirement for one strain to successfully invade and displace another. It is thought (unsure how widely?) that flu evolves antigenically, with new strains eventually displacing old ones, by mutating at key epitopes in ways that the immune system does not immediately pick up. That is, immune memory is biased to recall responses to conserved epitopes, which on average are probably less neutralizing. This will induce competition between mutant and resident viruses, but it would be symmetric, since infection with either would induce responses to conserved epitopes on the other. But if on infection with the mutant, immune memory sometimes reuses (boosts) antibody responses to target the mutated epitopes, those recycled antibodies might be less effective against the mutant, making the competition asymmetric.

    What this paper and Underwood (1980) suggest is that we can get this asymmetric, antibody-mediated competition fairly easily and without extensive memory. Underwood showed this more powerfully in rabbits, but in this paper too we see an indirect suggestion of asymmetry in relatively inexperienced children (Fig. 3). Mutants (future strains) successfully invade when they can trigger presumably recalled antibodies that are more harmful to the resident (soon historical) strain than the mutant. If this is so easy to do, as judged by the extensive data here, then it could be common.

    I've gone off on a theoretical limb here, but the paper is still important without these considerations. This work will be of interest to evolutionary biologists, epidemiologists, vaccinologists, and everyone else wondering what SARS-CoV-2 will do next and how immunity to antigenically variable pathogens works.

    We completely agree with the ideas mentioned above, and appreciate having it put in this nice context, particularly alongside the Underwood paper (with which we were not previously familiar). That said, we believe that the small number of recent children sera samples in the current study preclude us from drawing strong conclusions about the asymmetry--as the reviewer says, our data provides an indirect suggestion too. So overall we have not tried to expand this angle here because as the reviewer says, the paper is still important without these considerations. However, we are actively working to see if we can design a similar study with more children sera in the future to separately address the questions about asymmetry.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    An important question in coronavirology is what governs their ability to seemingly reinfect people regularly (within 2 or 3 years). While waning protective immunity has been proposed and is of current concern for SARS CoV-2, the role of antigenic drift driven by escape from neutralizing antibodies has not been well characterized. The authors have attempted to look at this through examining historical Spike proteins from HCoV-229E over a period of 30-odd years. The authors show that 229E evolves along a linear trajectory consistent with yearly selection by pre-existing immunity. Taking representative spike proteins from different time points into pseudovirus neut assays, they find that older spike proteins are less sensitive to neut by more recent sera. Conversely, spike proteins from prior to the birth of an individual display markedly less sensitivity to neut that those prevalent during the persons lifetime. Sequence analysis of the spike shows variation accruing in both N-termina regions and the RBD, parts of spike predominantly targeted by nABs. Lastly producing early spikes with chimeric RBDs from late viruses enhances the sensitivity to more recent sera.

    This is a potentially important MS that addresses a pertinent question that is of wide interest for the CoV2 pandemic. While it is limited in addressing the relative contribution of antigenic escape vs waning Ab titers because of the nature of the sample, the MS makes a strong case for Spike evolution being driven by antigenic escape.

    Thanks for the summary. We agree that our paper does not really address waning immunity because we don’t have sequential serum samples from the same individual. However, it does clearly show that antigenic evolution is important independent of waning immunity, because all of the experiments (e.g., Figure 2 and 3) show the same serum sample tested against newer spikes, and neutralization titers definitely decrease as the spike evolves. The reviewer is correct that this doesn’t rule out the possibility of waning immunity as a separate phenomenon, and we have been sure to emphasize that in the revised text.

    Reviewer #2 (Significance (Required)):

    While the Figs 1-3 are clear, the data in Fig 4 is somewhat preliminary. In all likelihood many people are making neutralizing antibodies both against RBD and the N-terminal region and the relative proportion probably underlies the variability in the data in Fig 4B. I think the MS would benefit from the following:

    A comparison of NTD vs RBD vs NTD/RBD chimeras in Fig 4B to give a fuller picture of antigenic escape with statistical support.

    The reviewer is correct that our manuscript does not provide a decisive answer on the relative role of NTD versus RBD targeting antibodies, although the data in Fig. 4B clearly show that RBD antibodies are important for many individuals as simply changing the RBD to that of newer viruses recapitulates the full spike antigenic evolution without any changes in the NTD or elsewhere (e.g., subject SD87_2 or SD85_3 in Fig 4B). However, for some other individuals NTD antibodies may play a role.

    In general, full dissection of the role of RBD versus NTD antibodies is beyond the scope of our study (and in some cases not even possible with the available volumes of the older serum). In any case, the major point of our study—the first experimental demonstration that seasonal coronaviruses undergo antigenic evolution—does not depend on dissecting the relative roles of RBD and NTD antibodies. We have therefore added new text explaining that we cannot fully parse the relative role of antibodies to these domains beyond knowing that RBD antibodies play n important role. We have added text to emphasize that antibodies to other regions including the NTD could also be important.

    A figure to map the polymorphic residues in Fig 4A onto the 229E spike structure to visualise their position and special relatedness, with perhaps a comparison with the latest knowledge of SASR CoV-2 epitopes.

    We agree that visualizing the variable sites on the structure is useful and have added such a visualization as a new panel in Figure 4. This allows us to more clearly show the clustering of variability in the RBD and NTD. This clustering of mutations in those regions is consistent with what is currently being seen with the emergence of SARS-CoV-2 variants with mutations in those regions of spike. However, given the divergence between SARS-CoV-2 and CoV-229E, we are not able to do a more fine-grained comparison of epitope sites as many important sites in the RBD and NTD do not have a clear one-to-one alignment (for instance, the RBD’s don’t even bind the same receptor).

    Additional discussion to reflect the new SARS CoV-2 variants and their potential selection by escape in the light of the authors data.

    We have updated the manuscript to describe the new SARS-CoV-2 variants (which mostly emerged after submission of our original manuscript) and how this emerging antigenic evolution of SARS-CoV-2 is consistent with what we saw in CoV-229E.

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    Referee #2

    Evidence, reproducibility and clarity

    An important question in coronavirology is what governs their ability to seemingly reinfect people regularly (within 2 or 3 years). While waning protective immunity has been proposed and is of current concern for SARS CoV-2, the role of antigenic drift driven by escape from neutralizing antibodies has not been well characterized. The authors have attempted to look at this through examining historical Spike proteins from HCoV-229E over a period of 30-odd years. The authors show that 229E evolves along a linear trajectory consistent with yearly selection by pre-existing immunity. Taking representative spike proteins from different time points into pseudovirus neut assays, they find that older spike proteins are less sensitive to neut by more recent sera. Conversely, spike proteins from prior to the birth of an individual display markedly less sensitivity to neut that those prevalent during the persons lifetime. Sequence analysis of the spike shows variation accruing in both N-termina regions and the RBD, parts of spike predominantly targeted by nABs. Lastly producing early spikes with chimeric RBDs from late viruses enhances the sensitivity to more recent sera.

    This is a potentially important MS that addresses a pertinent question that is of wide interest for the CoV2 pandemic. While it is limited in addressing the relative contribution of antigenic escape vs waning Ab titers because of the nature of the sample, the MS makes a strong case for Spike evolution being driven by antigenic escape.

    Significance

    While the Figs 1-3 are clear, the data in Fig 4 is somewhat preliminary. In all likelihood many people are making neutralizing antibodies both against RBD and the N-terminal region and the relative proportion probably underlies the variability in the data in Fig 4B. I think the MS would benefit from the following:

    • A comparison of NTD vs RBD vs NTD/RBD chimeras in Fig 4B to give a fuller picture of antigenic escape with statistical support.

    • A figure to map the polymorphic residues in Fig 4A onto the 229E spike structure to visualise their position and special relatedness, with perhaps a comparison with the latest knowledge of SASR CoV-2 epitopes.

    • Additional discussion to reflect the new SARS CoV-2 variants and their potential selection by escape in the light of the authors data.

    Read the original source
    Was this evaluation helpful?
  3. Reviewed by Review Commons

    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

    The authors investigated the cross-neutralization capacity of serum antibodies to past and future 229E coronaviruses using 229E spikes isolated from five time points and sera from two different periods (1985-1995 and 2020). They demonstrated a general pattern of asymmetric cross-neutralization, with sera cross-reactive to historical but not future strains. Using chimeras, the authors showed this pattern was mostly driven by antibodies to the evolving RBD. The rate of change in the neutralization titer, a possible measure of antigenic evolution, was estimated to be on par with that of flu B viruses. Interesting differences in individuals' cross-neutralization capacity were observed. The main take-away is that reinfection with 229E is enabled by antigenic escape, not "weak" immunity after infection (as proposed by others).

    Major comments:

    The key conclusions are convincing and justified by the data. The work is clearly presented and presented with sufficient detail for reproducibility. Characteristically and laudably, the authors have made all the code and data publicly available on GitHub.

    Minor comments:

    p. 3: Perhaps it is clearer to write that 229E has been identified/isolated in humans for >50 y? Or do you really mean to imply (by contrast with "circulated") that NL63 emerged very recently?

    p. 3: An important citation for the antigenic implications of the ladder-like phylogeny AND phylogenetic clustering by date is the classic paper introducing phylodynamics by Grenfell et al. (2004, Science).

    p. 4: I might not be like all readers, but I prefer to see a bit in the main text about the source of sera for this kind of study. (I wonder about age, if donors are healthy, etc.)

    p. 4: "Our reason for focusing..." stops short. Is the idea that these are probably people who were recently infected?

    p. 5: Probably my biggest suggestion for the paper is that it mention another relevant study. In 1980, Anne Underwood demonstrated similar asymmetric cross-immunity among early strains of H3N2 (but using rabbits, not human sera), finding that antibodies raised to one strain reacted more strongly by HAI to past strains than to later strains (doi: 10.1128/IAI.27.2.397-404.1980). This relates to the significance of the paper (next section).

    Obviously, there are citations to update throughout due to the booming SARS-CoV-2 literature.

    Significance

    This study, if anything, undersells itself. Obviously it is a huge contribution to our understanding of how a seasonal coronavirus that bears important phenotypic resemblance to SARS-CoV-2 evolves, but I think it is also providing a foundational piece of evidence--a mechanism--of how rapid viral turnover (by antigenic evolution) occurs. There is no reason to think this should be limited to the coronaviruses, and I suspect the evidence here will go a long way to unifying the evolutionary and epidemiological dynamics of fast-evolving viruses.

    Asymmetric competition is nearly an ecological requirement for one strain to successfully invade and displace another. It is thought (unsure how widely?) that flu evolves antigenically, with new strains eventually displacing old ones, by mutating at key epitopes in ways that the immune system does not immediately pick up. That is, immune memory is biased to recall responses to conserved epitopes, which on average are probably less neutralizing. This will induce competition between mutant and resident viruses, but it would be symmetric, since infection with either would induce responses to conserved epitopes on the other. But if on infection with the mutant, immune memory sometimes reuses (boosts) antibody responses to target the mutated epitopes, those recycled antibodies might be less effective against the mutant, making the competition asymmetric.

    What this paper and Underwood (1980) suggest is that we can get this asymmetric, antibody-mediated competition fairly easily and without extensive memory. Underwood showed this more powerfully in rabbits, but in this paper too we see an indirect suggestion of asymmetry in relatively inexperienced children (Fig. 3). Mutants (future strains) successfully invade when they can trigger presumably recalled antibodies that are more harmful to the resident (soon historical) strain than the mutant. If this is so easy to do, as judged by the extensive data here, then it could be common.

    I've gone off on a theoretical limb here, but the paper is still important without these considerations. This work will be of interest to evolutionary biologists, epidemiologists, vaccinologists, and everyone else wondering what SARS-CoV-2 will do next and how immunity to antigenically variable pathogens works.

    Read the original source
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