A human coronavirus evolves antigenically to escape antibody immunity

  1. Rachel T. Eguia
  2. Katharine H. D. Crawford
  3. Terry Stevens-Ayers
  4. Laurel Kelnhofer-Millevolte
  5. Alexander L. Greninger
  6. Janet A. Englund
  7. Michael J. Boeckh
  8. Jesse D. Bloom

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Abstract

There is intense interest in antibody immunity to coronaviruses. However, it is unknown if coronaviruses evolve to escape such immunity, and if so, how rapidly. Here we address this question by characterizing the historical evolution of human coronavirus 229E. We identify human sera from the 1980s and 1990s that have neutralizing titers against contemporaneous 229E that are comparable to the anti-SARS-CoV-2 titers induced by SARS-CoV-2 infection or vaccination. We test these sera against 229E strains isolated after sera collection, and find that neutralizing titers are lower against these “future” viruses. In some cases, sera that neutralize contemporaneous 229E viral strains with titers >1:100 do not detectably neutralize strains isolated 8–17 years later. The decreased neutralization of “future” viruses is due to antigenic evolution of the viral spike, especially in the receptor-binding domain. If these results extrapolate to other coronaviruses, then it may be advisable to periodically update SARS-CoV-2 vaccines.

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  1. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/4768672.

    We, the students of MICI5029/5049, a Graduate Level Molecular Pathogenesis Journal Club at Dalhousie University in Halifax, NS, Canada, hereby submit a review of the following BioRxiv preprint:

    Rachel Eguia, Katharine H. D. Crawford, Terry Stevens-Ayers, Laurel Kelnhofer-Millevolte, Alexander L. Greninger, Janet A. Englund, Michael J. Boeckh, Jesse D. Bloom. A human coronavirus evolves antigenically to escape antibody immunity. bioRxiv 2020.12.17.423313; doi:https://doi.org/10.1101/2020.12.17.423313

    We will adhere to the Universal Principled (UP) Review guidelines proposed in:

    Universal Principled Review: A Community-Driven Method to Improve Peer Review. Krummel M, Blish C, Kuhns M, Cadwell K, Oberst A, Goldrath A, Ansel KM, Chi H, O'Connell R, Wherry EJ, Pepper M; Future Immunology Consortium. Cell. 2019 Dec 12;179(7):1441-1445. doi: 10.1016/j.cell.2019.11.029.

    SUMMARY:  Egula et al. investigated the ability of human sera collected over decades to neutralize contemporary and non-contemporary strains of HCoV-229E (229E). Using sera and viruses collected from the late 1980's to the present, the authors found that human sera from 1982 was unable to neutralize subsequent generations of 229E viruses, whereas sera recently collected from patients alive and likely exposed to 229E strains from 1982 could neutralize both modern and historic viruses, thereby providing some evidence for durable immunity. Finally, the authors confirmed that most of the mutations that prevent neutralization of modern viruses were found in the 3 loops of the receptor binding domain, although mutations in the N-terminal domain also likely contribute to antigenic escape.

    OVERALL ASSESSMENT: This is a good article with solid experiments and conclusions. We appreciate the way the article was written; it is clear and concise and follows a sound logical progression. However, we are not convinced that the work is particularly novel, although this may be an issue of framing (see final section on Subjective Criteria, below).

    STRENGTHS: The manuscript is well written, with a logical flow of ideas. Bioinformatics and neutralization data appear to be robust. Overall, while we have some concerns about statistics, the data is convincing.

    WEAKNESSES: The narrowing of sera samples for analysis seems to limit the scope of the study, and we think it might bias the results. Because of this, we are unsure if this data can be generalized to a larger population, or if it is only applicable to those whose antibody responses mirror the cutoffs chosen in this study. 

    DETAILED U.P. ASSESSMENT:

    OBJECTIVE CRITERIA (QUALITY)

    1.  Quality: Experiments (1–3 scale) SCORE = 1.5

    ●       Figure by figure, do experiments, as performed, have the proper controls?

    -        Fig. 1A: Since the authors have identified regions for each strain, they should justify relevance of those they selected compared to the region from which the sera they are testing are coming from. For instance, is it likely that the individual's sera tested would be exposed to viruses from Australia, China and the USA and if not, how would the phylogenetic divergence affect neutralization studies? It would have been helpful if the authors had also compared neutralization between closely related viruses from the same regions to determine if the sera was better at neutralizing certain spikes than others they selected. 

    o    Furthermore, for the figure legend, the distance bar labelled 4 years is not described in the figure legend and this would help the reader follow along.

    -        Fig. 2C: The fold change representation of neutralization titers has a lot of variance and is difficult to determine any overall result from this figure. 

    -        Fig. 4B: The authors should have followed this experiment using NTD chimeras, as changes in the NTD of Spike may have clarified why, for some individuals, the RBD chimera had higher neutralization responses than the full Spike. 

    o    Furthermore, without stats on this figure how can the authors claim that "neutralization activity was rapidly eroded by antigenic evolution" considering that some of the sera did not see change. This is not a convincing claim without statistics.

    ●       Are specific analyses performed using methods that are consistent with answering the specific question?

    -        Fig. 2: There are no stats performed for neutralization titers. Is it common practice in the field to conduct only one replicate for these assays? Conclusions could be strengthened by studying multiple replicates (N=3) and applying appropriate statistical tests.  

    ●       Is there the appropriate technical expertise in the collection and analysis of data presented?

    -        Yes.

    ●       Do analyses use the best-possible (most unambiguous) available methods quantified via appropriate statistical comparisons?

    -        Yes.

    ●       Are controls or experimental foundations consistent with established findings in the field? A review that raises concerns regarding inconsistency with widely reproduced observations should list at least two examples in the literature of such results. Addressing this question may occasionally require a supplemental figure that, for example, re-graphs multi-axis data from the primary figure using established axes or gating strategies to demonstrate how results in this paper line up with established understandings. It should not be necessary to defend exactly why these may be different from established truths, although doing so may increase the impact of the study and discussion of discrepancies is an important aspect of scholarship.

    -        Yes.

     

    2. Quality: Completeness (1–3 scale) SCORE = 1

    ●       Does the collection of experiments and associated analysis of data support the proposed title- and abstract-level conclusions? Typically, the major (title- or abstract-level) conclusions are expected to be supported by at least two experimental systems,

    -        Yes, the conclusions are well supported by three experimental systems: evolutionary analysis, human sera neutralization assays, and chimera spike receptor neutralization analysis.

    ●       Are there experiments or analyses that have not been performed but if ''true'' would disprove the conclusion (sometimes considered a fatal flaw in the study)? In some cases, a reviewer may propose an alternative conclusion and abstract that is clearly defensible with the experiments as presented, and one solution to ''completeness'' here should always be to temper an abstract or remove a conclusion and to discuss this alternative in the discussion section.

    -        There is no positive control for the neutralization assays. It would be beneficial to show the reader that these assays perform as expected against known antibody/antigen combinations. This would show the reader that the authors know what to expect from their other neutralization assays.

    3. Quality: Reproducibility (1–3 scale) SCORE = 2

    ●       Figure by figure, were experiments repeated per a standard of 3 repeats or 5 mice per cohort, etc.?

    -        Repeats were not performed for the neutralization assays. 

    ●       Is there sufficient raw data presented to assess rigor of the analysis?

    -        Yes.

    ●       Are methods for experimentation and analysis adequately outlined to permit reproducibility?

    -        Yes.

    ●       If a ''discovery'' dataset is used, has a ''validation'' cohort been assessed and/or has the issue of false discovery been addressed?

    -        N/A.

     

    4. Quality: Scholarship (1–4 scale but generally not the basis for acceptance or rejection) SCORE = 1.5

    ●   Has the author cited and discussed the merits of the relevant data that would argue against their conclusion?

    -        Is it possible that the decrease seen in the older patients for the neutralization of historic viruses could be related to immune senescence or some immune amnesia-like phenotype from a different infection? This might be worth exploring in the discussion as it is an interesting data point.

     

    ●   Has the author cited and/or discussed the important works that are consistent with their conclusion and that a reader should be especially familiar when considering the work?

    -        Yes, the authors did a wonderful job explaining that there is little work in this area and highlighting why they believe their work is novel.

     

    ●   Specific (helpful) comments on grammar, diction, paper structure, or data presentation (e.g., change a graph style or color scheme) go in this section, but scores in this area should not to be significant bases for decisions.

    -        Fig. 2: box plot could perhaps be formatted differently. As it is shown, it seems to work against the authors conclusions. Also, were stats performed on this box plot? If they are, they should be shown. Stats would make the plot more understandable in context of your other data.

     

    MORE SUBJECTIVE CRITERIA (IMPACT)

    1.  Impact: Novelty/Fundamental and Broad Interest (1–4 scale) SCORE = 2

    ●   A score here should be accompanied by a statement delineating the most interesting and/or important conceptual finding(s), as they stand right now with the current scope of the paper. A ''1'' would be expected to be understood for the importance by a layperson but would also be of top interest (have lasting impact) on the field.

    -        This article is interesting and provoked a great deal of discussion in our journal club. It advances our current understanding of antigen evasion by viruses (specifically coronaviruses) and our immune system's ability to detect and neutralize historic coronaviruses. It also confirms that coronaviruses mutate largely in the receptor binding domains, but not as much elsewhere, to evade the immune system.

    -        However, we have not awarded full points on novelty because on some level we think that the results are to be expected. People who have been exposed to a virus likely have neutralizing antibodies against them. People who haven't likely do not. Because this takes up most of Figures 2 and 3, we believe this is a major conclusion of the paper.

    -        With that said, this remains foundational work for understanding human serum neutralization capacity for coronaviruses over time. A greater emphasis on the implications for durable anti-coronavirus immunity in the Discussion and elsewhere may strengthen the perception of novelty.  

     

    ●   How big of an advance would you consider the findings to be if fully supported but not extended? It would be appropriate to cite literature to provide context for evaluating the advance. However, great care must be taken to avoid exaggerating what is known comparing these findings to the current dogma (see Box 2). Citations (figure by figure) are essential here. 

    o    N/A

     

    2.  Impact: Extensibility (1–4 or N/A scale) SCORE = N/A

    ●  Has an initial result (e.g., of a paradigm in a cell line) been extended to be shown (or implicated) to be important in a bigger scheme (e.g., in animals or in a human cohort)?

    ●  This criterion is only valuable as a scoring parameter if it is present, indicated by the N/A option if it simply doesn't apply. The extent to which this is necessary for a result to be considered of value is important. It should be explicitly discussed by a reviewer why it would be required. What work (scope and expected time) and/or discussion would improve this score, and what would this improvement add to the conclusions of the study? Care should be taken to avoid casually suggesting experiments of great cost (e.g., ''repeat a mouse-based experiment in humans'') and difficulty that merely confirm but do not extend (see Bad Behaviors, Box 2).

    o    N/A

  2. Version published to 10.1371/journal.ppat.1009453
  3. ScreenIT

    SciScore for 10.1101/2020.12.17.423313: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: These sera were obtained from the Infectious Disease Sciences Biospecimen Repository at the Vaccine and Infectious Disease Division (VIDD) of the Fred Hutchinson Cancer Research Center in Seattle, WA, and were collected from prospective bone marrow donors with approval from the Human Subjects Institutional Review Board.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    To mitigate any possible well-to-well differences in transfection efficiency in a 96-well plate format, we first bulk transfected a dish of 293T cells, followed by seeding the 96-well plates routinely used in neutralization assays and viral titering.
    293T
    suggested: None
    These viral dilutions were made in TC-treated “set-up” 96-well plates and then transferred to the pre-seeded 293T-ACE2-TMPRSS2 cells from the previous day.
    293T-ACE2-TMPRSS2
    suggested: None
    Software and Algorithms
    SentencesResources
    We parsed full-length human-isolate spikes encoding unique proteins from this sequence set (see https://github.com/jbloomlab/CoV_229E_antigenic_drift/blob/master/results/get_parse_spikes.md), used mafft (Katoh and Standley, 2013) to align the protein sequences, and used a custom Python script (https://github.com/jbloomlab/CoV_229E_antigenic_drift/blob/master/prot_to_codon_alignment.py) to build a codon alignment from the protein alignment (File S1).
    Python
    suggested: (IPython, RRID:SCR_001658)
    The phylogenetic tree topology was inferred using IQ-TREE (Minh et al., 2020) using a codon-substitution model (Muse and Gaut, 1994) with a transition-transversion ratio and F3X4 empirical codon frequencies.
    IQ-TREE
    suggested: (IQ-TREE, RRID:SCR_017254)

    Results from OddPub: Thank you for sharing your code and data.

    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.

    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.

  4. Review Commons

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

    Learn more at Review Commons

    Reply to the reviewers

    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.

  5. Review Commons

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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.

  6. 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.

  7. ScreenIT

    SciScore for 10.1101/2020.12.17.423313: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementThese sera were obtained from the Infectious Disease​ Sciences Biospecimen Repository at the Vaccine and Infectious Disease Division (VIDD) of the Fred Hutchinson Cancer Research Center in Seattle, WA, and were collected from prospective bone marrow donors with approval from the Human Subjects Institutional Review Board​.Randomizationnot detected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variablenot detected.Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Specifically, an approximately 90% confluent 10 cm dish of 293T cells was transfected with 8.5 µg APN-expressing plasmid, 1 µg of TMPRSS2-expressing plasmid, and 0.5 µg of carrier DNA (Promega, E4881) to achieve an 8.5:1 ratio of APN:TMPRSS2.
    293T
    suggested: None
    These viral dilutions were made in TC-treated “set-up” 96-well plates and then transferred to the pre-seeded 293T-ACE2-TMPRSS2 cells from the previous day.
    293T-ACE2-TMPRSS2
    suggested: None
    Software and Algorithms
    SentencesResources
    We parsed full-length human-isolate spikes encoding unique proteins from this sequence set (see https://github.com/jbloomlab/CoV_229E_antigenic_drift/blob/master/results/get_parse_spikes.md​), used mafft​ (Katoh and Standley, 2013) to align the protein sequences, and used a custom Python script (​https://github.com/jbloomlab/CoV_229E_antigenic_drift/blob/master/prot_to_codon_alignment.py​) to build a codon alignment from the protein alignment (File S1).
    Python
    suggested: (IPython, RRID:SCR_001658)
    The phylogenetic tree topology was inferred using IQ-TREE (Minh et al., 2020) using a codon-substitution model (Muse and Gaut, 1994) with a transition-transversion ratio and F3X4 empirical codon frequencies.
    IQ-TREE
    suggested: (IQ-TREE, RRID:SCR_017254)

    Results from OddPub: Thank you for sharing your data.

    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.

    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.

  8. Version published to 10.1101/2020.12.17.423313v1 on bioRxiv