Identification of a conserved neutralizing epitope present on spike proteins from all highly pathogenic coronaviruses

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    Silva and colleagues present useful findings related to the isolation of an anti-S2 antibody that recognizes a previously uncharacterized SARS-CoV2 Spike (S) epitope, adding to the growing repertoire of anti-S antibodies that broadly cross-react against human and zoonotic coronaviruses. The evidence supporting the claims of the authors is solid, although antibody effectiveness as a prophylactic or therapeutic reagent in an animal model would have strengthened the study. The work will be of interest to biologists working to develop pan-coronavirus therapies.

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Abstract

Three pathogenic human coronaviruses have emerged within the last 20 years, with SARS-CoV-2 causing a global pandemic. Although therapeutic antibodies targeting the SARS-CoV-2 spike currently focus on the poorly conserved receptor-binding domain, targeting essential neutralizing epitopes on the more conserved S2 domain may provide broader protection. We report an antibody binding an epitope conserved in the pre-fusion core of MERS-CoV, SARS-CoV and SARS-CoV-2 spike S2 domains. Antibody 3A3 binds a conformational epitope with ~2.5 nM affinity and neutralizes spike from SARS-CoV, SARS-CoV-2 and variants of concern in in vitro pseudovirus assays. Hydrogen-deuterium exchange mass spectrometry identified residues 980-1006 in the flexible hinge region at the S2 apex as the 3A3 epitope, suggesting 3A3 prevents the S2 conformational rearrangements required for conversion to the spike post-fusion state and virus-host cell fusion. This work defines a conserved vulnerable site on the SARS-CoV-2 S2 domain and guides the design of pan-protective spike immunogens.

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  1. Author Response

    Reviewer #1 (Public Review):

    The manuscript by Silva et al. "Evaluation of the highly conserved S2 hairpin hinge as a pan-coronavirus target" seeks to evaluate a new epitope target on the S2 domain of SARS-CoV2 Spike protein and evaluate its potential as a pan-coronavirus target. This is an impressive combination of extensive structural, HDXMS-based dynamics and antibody engineering approaches. What is missing is a detailed correlation of HDXMS with Spike dynamics. The authors have not examined the allosteric effects of 3A3 binding to the Spike trimer, specifically cooperativity in antibody binding. Does binding of one Fab positively or negatively impact the subsequent binding of antibody? In this regard, readers would benefit from HDXMS spectral envelopes in figures, at least for the epitope locus peptides. Further, what is the effect of the intrinsic ensemble behavior of the Spike protein on 3A3 interactions? In a broader sense antibody binding is assisted by intrinsic trimer ensemble behavior, as observed by the lowered binding to the omicron variant- but are there induced binding effects? It would help to better integrate HDXMS with cryo-EM and antibody engineering. It is a novel, less explored epitope target on the S2 domain. Overall, a more definitive mechanistic conclusion for how targeting the S2 hinge can advance future pan-coronavirus strategies is missing.

    1. Given that the authors have demonstrated ensemble switching behavior from 4 ℃ to 37 ℃ (Costello et al. (2021)) why is this not factored in how the HDXMS is carried out? The samples were stored, frozen at -80 ℃, thawed, and equilibrated for 20 min at 20 ℃ with or without antibody present and analyzed by HDXMS. However, the reported t1/2 for trimer tightening at 37 ℃ is t1/2 = 2.5 h (Supplementary Fig. 7). The samples should ideally be analyzed under standardized conditions with the stable conformer. Sample heterogeneity from HDXMS is likely due to any of the following contributing factors:

    i) Intrinsic ensemble heterogeneity (Costello et al. (2021)), Kinetics of RBD- up and down conformational switching

    ii) Cooperativity of Fab binding.

    iii) Partial occupancy of trimer epitopes with bivalent IgG.

    iv) Combination of cooperativity effects and partial binding effects

    I would predict for any of the above reasons, it is intriguing why are there no bimodal kinetics of deuterium exchange reported. Partial occupancy should be evident from HDXMS paratope analysis.

    1. Pan-coronavirus neutralization potential is clearly evident. It is intriguing that the antibodies were isolated after immunization with an authentic MERS S2 domain but showed better selectivity to full-length 6P-engineered Spike. How is cooperativity built into antibody binding, given that the epitope site is occluded to various extents by the S1 domain and access is contingent upon RBD up-down kinetics?
    1. I am surprised that there is no allostery described for 3A3 (Supplementary figures 5, 6).

    The HDX-MS experiments presented in this work were carried out by the D’Arcy lab and published in a preprint on bioRxiv (originally posted on February 1, 2021) prior to publication of Costello et al. (first posted to bioRxiv July 11, 2021, epub March 2, 2022). Indeed, our bioRxiv posting inspired the Marqusee lab to request 3A3 for inclusion in their work focused on the conformational heterogeneity of the spike protein. Without prior knowledge of the conformational heterogeneity, we carried out these epitope mapping experiments at 25Ç, which allowed us to successfully mapped the epitope without determining which conformation the antibody prefers.

    The data presented in Costello et al. further confirms the location of 3A3’s epitope presented here and provides additional information about its preference for different conformational states within the spike protein. We have included an additional comment in the methods section (lines 660-661) stating, “The location of the 3A3 epitope was confirmed in a separate experiment carried out over the temperature range of 4 to 37 °C (Costello et al. 2022).”

    This is a clear example of the value of pre-prints to stimulate timely scientific collaboration. While Costello et al. used 3A3 as a tool to probe spike dynamics, here we highlight the original work that identified the epitope.

    Spectral envelopes have been provided (Supplementary Fig. 4b and Supplementary Table 3).

    The HDX-MS data provides limited insight into possible cooperative or allosteric binding of the 3A3 antibody because of other sources of heterogeneity such as spike dynamics and partial occupancy of the spike epitopes. However, no difference in occupancy was detected when HDX-MS with 3A3 Fab was compared to the same experiment with bivalent 3A3 IgG. It should be noted that in this HDX system, the antibody is not bound so tightly that the spectra are bimodal, showing the exchange of bound and unbound populations separately. Though HDX-MS experiments were performed in slight Fab or IgG excess of 1:1 Fab:spike monomer stoichiometry, the absolute stoichiometry in the context of the spike trimer is unclear.

    Reviewer #2 (Public Review):

    The authors report a conserved spike S2 hinge epitopes and two conformationally selective antibodies that help elucidate spike behavior. This work defines a third class of S2 antibody and provides insights into the potency and limitations of targeting this S2 epitope for future pan-coronavirus strategies.

    Thank you for your review of this manuscript.

    Reviewer #3 (Public Review):

    The study by Silva et al details the discovery and evaluation of a third class of broadly cross-reactive anti-Spike antibody that binds a conserved hinge region in the S2 domain. After immunizing mice with a stabilized S2 protein from MERS and generating scFv phage libraries, the authors were able to identify antibody 3A3, which showed broad cross-reactivity with SARS2 (including Omicron BA.1), SARS1, MERS, and HKU1 spike proteins. Using a combination of a low-resolution cryo-EM structure and HDX mass spectrometry, the authors were able to map amino acids in the antibody paratope and spike epitope, the latter of which is the hinge region of the Spike S2 domain (residues 980-1005) that plays a critical role in pre- to -post-fusion conformational changes. Through well-executed and comprehensive mutagenesis, binding, and functional assays, the authors further validated critical residues that lead to antibody escape, which centered around the 2P residues and diminished viral entry. While 3A3 and an affinity-enhanced engineered version, RAY53, did not show potent in vitro neutralization against the authentic virus, the antibody was shown to recruit Fc effector functions for viral clearance, in vitro.

    Overall, the conclusions of this paper are well supported by the data, but the usefulness of such antibodies is likely limited. The work can be strengthened by extending the analysis of 3A3-like antibodies in the context of human immune responses and in vivo effectiveness.

    1. Isolation of 3A3 was achieved after the generation of scFv-phage libraries following immunization with a MERS S2-domain immunogen in a mouse model. The fact that 3A3 binds well to 2P-stabilized sequences and binding/neutralization is diminished upon reversion of 2P mutations back to the native spike sequence (Figures 3a, 4c, and 5b), suggest that such antibodies would likely not arise from natural infection. This contrasts the isolation of fusion peptide and stem helix-directed antibodies, which were isolated from both immunized animals and convalescent individuals. To make their results more solid regarding the use of such antibodies in future vaccine strategies, the authors should provide evidence that 3A3-like antibodies can be identified in human donors. For example, they could enrich donor-derived S2-specific antibodies that bind both MERS and SARS2 S2 domains and evaluate the fraction of antibodies that recognize the hinge-epitope using competition binding assays (either ELISA or BLI), which have commonly been used to map epitope-specific sera responses. This could also be achieved with nsEMPEM of polyclonal IgGs bound to S2 proteins.
    1. The authors speculate in the discussion that strategies to enhance access to the hinge epitope, which may include ACE2-mimicking antibodies, could promote enhanced viral clearance. In addition to ACE2-mimicking antibodies, several antibodies have been described that bind the RBD and promote S1 shedding (see for instance mAb S2A4 - Piccoli et al, 2020, Cell). Several 2nd generation vaccine platforms utilize RBD-only immunogens that are likely to induce high titers of ACE2-mimicking and cross-reactive S1-shedding antibodies. Thus, adding in vitro neutralization and ADCC experiments to assess synergy between 3A3/RAY53 and such antibodies would booster this speculative claim and be of interest to many in the field developing strategies for pan-coronavirus therapies.
    1. The authors provide in vitro evidence in Figure 5c,d for Fc-mediated viral clearance. While in vivo data to show effectiveness in animal models is ideal, additional in vitro data that utilize engineered constructs that modulate effector function (e.g., DLE (+) or LALA (-)) would boost the authors' claims regarding Fc-mediated viral clearance mechanisms by 3A3/RAY53.
    1. Though we do not plan to isolate 3A3-like antibodies from human donors, there is evidence that these antibodies are elicited in infected humans via analysis of polyclonal responses in Claireaux et al 2022. We also know of several studies on naturally occurring S2 hinge targeting antibodies from colleagues that are in preparation. Understanding the therapeutic role of this antibody class is relevant to the study of broadly-reactive S2 antibodies, even if that role is limited.

    2. We agree that synergy between S2 hinge epitope binding antibodies and ACE2 mimicking antibodies will be very interesting to investigate. We hope to pursue this in future work.

    3. We agree these are excellent controls to include, in addition to isotype controls already shown. In accordance with the eLife COVID research policy, we minimized our claims around Fc-effector functions elicited by RAY53 and stated that further experiments to confirm our preliminary findings are needed.

    The existing description of the effector function experiments states in lines 392-392 “These results indicate that RAY53 binding is compatible with ADCP and ADCC,” which is already a very limited claim.

    We also added in line 450 that S2 core-binding antibodies “require further validation” of their ability to recruit effector functions.

    We appreciate the importance of controls providing effector function modulation and will include the LALAPG mutations as a standard component of our future ADCC evaluation. However, given our focus on the relevance of the epitope and consistency of the Fc regions across the antibodies, we felt that the isotype and positive control antibodies (target binding controls) were the most relevant controls to include in this study.

  2. eLife assessment

    Silva and colleagues present useful findings related to the isolation of an anti-S2 antibody that recognizes a previously uncharacterized SARS-CoV2 Spike (S) epitope, adding to the growing repertoire of anti-S antibodies that broadly cross-react against human and zoonotic coronaviruses. The evidence supporting the claims of the authors is solid, although antibody effectiveness as a prophylactic or therapeutic reagent in an animal model would have strengthened the study. The work will be of interest to biologists working to develop pan-coronavirus therapies.

  3. Reviewer #1 (Public Review):

    The manuscript by Silva et al. "Evaluation of the highly conserved S2 hairpin hinge as a pan-coronavirus target" seeks to evaluate a new epitope target on the S2 domain of SARS-CoV2 Spike protein and evaluate its potential as a pan-coronavirus target. This is an impressive combination of extensive structural, HDXMS-based dynamics and antibody engineering approaches. What is missing is a detailed correlation of HDXMS with Spike dynamics. The authors have not examined the allosteric effects of 3A3 binding to the Spike trimer, specifically cooperativity in antibody binding. Does binding of one Fab positively or negatively impact the subsequent binding of antibody? In this regard, readers would benefit from HDXMS spectral envelopes in figures, at least for the epitope locus peptides. Further, what is the effect of the intrinsic ensemble behavior of the Spike protein on 3A3 interactions? In a broader sense antibody binding is assisted by intrinsic trimer ensemble behavior, as observed by the lowered binding to the omicron variant- but are there induced binding effects? It would help to better integrate HDXMS with cryo-EM and antibody engineering. It is a novel, less explored epitope target on the S2 domain. Overall, a more definitive mechanistic conclusion for how targeting the S2 hinge can advance future pan-coronavirus strategies is missing.

    Major Comments:

    1. Given that the authors have demonstrated ensemble switching behavior from 4 ℃ to 37 ℃ (Costello et al. (2021)) why is this not factored in how the HDXMS is carried out? The samples were stored, frozen at -80 ℃, thawed, and equilibrated for 20 min at 20 ℃ with or without antibody present and analyzed by HDXMS. However, the reported t1/2 for trimer tightening at 37 ℃ is t1/2 = 2.5 h (Supplementary Fig. 7). The samples should ideally be analyzed under standardized conditions with the stable conformer. Sample heterogeneity from HDXMS is likely due to any of the following contributing factors:
      i) Intrinsic ensemble heterogeneity (Costello et al. (2021)), Kinetics of RBD- up and down conformational switching
      ii) Cooperativity of Fab binding.
      iii) Partial occupancy of trimer epitopes with bivalent IgG.
      iv) Combination of cooperativity effects and partial binding effects

    I would predict for any of the above reasons, it is intriguing why are there no bimodal kinetics of deuterium exchange reported. Partial occupancy should be evident from HDXMS paratope analysis.

    1. Pan-coronavirus neutralization potential is clearly evident. It is intriguing that the antibodies were isolated after immunization with an authentic MERS S2 domain but showed better selectivity to full-length 6P-engineered Spike. How is cooperativity built into antibody binding, given that the epitope site is occluded to various extents by the S1 domain and access is contingent upon RBD up-down kinetics?

    2. I am surprised that there is no allostery described for 3A3 (Supplementary figures 5, 6).

  4. Reviewer #2 (Public Review):

    The authors report a conserved spike S2 hinge epitopes and two conformationally selective antibodies that help elucidate spike behavior. This work defines a third class of S2 antibody and provides insights into the potency and limitations of targeting this S2 epitope for future pan-coronavirus strategies.

  5. Reviewer #3 (Public Review):

    The study by Silva et al details the discovery and evaluation of a third class of broadly cross-reactive anti-Spike antibody that binds a conserved hinge region in the S2 domain. After immunizing mice with a stabilized S2 protein from MERS and generating scFv phage libraries, the authors were able to identify antibody 3A3, which showed broad cross-reactivity with SARS2 (including Omicron BA.1), SARS1, MERS, and HKU1 spike proteins. Using a combination of a low-resolution cryo-EM structure and HDX mass spectrometry, the authors were able to map amino acids in the antibody paratope and spike epitope, the latter of which is the hinge region of the Spike S2 domain (residues 980-1005) that plays a critical role in pre- to -post-fusion conformational changes. Through well-executed and comprehensive mutagenesis, binding, and functional assays, the authors further validated critical residues that lead to antibody escape, which centered around the 2P residues and diminished viral entry. While 3A3 and an affinity-enhanced engineered version, RAY53, did not show potent in vitro neutralization against the authentic virus, the antibody was shown to recruit Fc effector functions for viral clearance, in vitro.

    Overall, the conclusions of this paper are well supported by the data, but the usefulness of such antibodies is likely limited. The work can be strengthened by extending the analysis of 3A3-like antibodies in the context of human immune responses and in vivo effectiveness.

    1. Isolation of 3A3 was achieved after the generation of scFv-phage libraries following immunization with a MERS S2-domain immunogen in a mouse model. The fact that 3A3 binds well to 2P-stabilized sequences and binding/neutralization is diminished upon reversion of 2P mutations back to the native spike sequence (Figures 3a, 4c, and 5b), suggest that such antibodies would likely not arise from natural infection. This contrasts the isolation of fusion peptide and stem helix-directed antibodies, which were isolated from both immunized animals and convalescent individuals. To make their results more solid regarding the use of such antibodies in future vaccine strategies, the authors should provide evidence that 3A3-like antibodies can be identified in human donors. For example, they could enrich donor-derived S2-specific antibodies that bind both MERS and SARS2 S2 domains and evaluate the fraction of antibodies that recognize the hinge-epitope using competition binding assays (either ELISA or BLI), which have commonly been used to map epitope-specific sera responses. This could also be achieved with nsEMPEM of polyclonal IgGs bound to S2 proteins.

    2. The authors speculate in the discussion that strategies to enhance access to the hinge epitope, which may include ACE2-mimicking antibodies, could promote enhanced viral clearance. In addition to ACE2-mimicking antibodies, several antibodies have been described that bind the RBD and promote S1 shedding (see for instance mAb S2A4 - Piccoli et al, 2020, Cell). Several 2nd generation vaccine platforms utilize RBD-only immunogens that are likely to induce high titers of ACE2-mimicking and cross-reactive S1-shedding antibodies. Thus, adding in vitro neutralization and ADCC experiments to assess synergy between 3A3/RAY53 and such antibodies would booster this speculative claim and be of interest to many in the field developing strategies for pan-coronavirus therapies.

    3. The authors provide in vitro evidence in Figure 5c,d for Fc-mediated viral clearance. While in vivo data to show effectiveness in animal models is ideal, additional in vitro data that utilize engineered constructs that modulate effector function (e.g., DLE (+) or LALA (-)) would boost the authors' claims regarding Fc-mediated viral clearance mechanisms by EA3/RAY53.

  6. SciScore for 10.1101/2021.01.31.428824: (What is this?)

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

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Antibodies
    SentencesResources
    Four rounds of panning were used to isolate scFvs binding both MERS S2 and SARS-2 spike using the following solutions coated on high binding plates: 2 μg/ml anti-c-myc tag antibody (Invitrogen) to eliminate phage expressing no or truncated scFv (Round 1), 2 μg/ml MERS S2 (Round 2), 2 μg/ml SARS-2 spike (Round 3), and 0.4 μg/ml SARS-2 spike (Round 4).
    anti-c-myc tag
    suggested: None
    Duplicate serial dilutions of each full-length antibody were allowed to bind each coat, and the secondary antibody solution was a 1:1200 dilution of goat-anti-human IgG Fc-HRP (SouthernBiotech).
    IgG Fc-HRP ( SouthernBiotech) .
    suggested: None
    Western blot of antibody binding to coronavirus spike proteins: Purified coronavirus spike proteins (SARS-2 HexaPro, SARS-2, MERS, and HKU1) were reduced and boiled, and 50 ng of each was subjected to SDS-PAGE and transfer to PVDF membranes in quadruplicate.
    HKU1
    suggested: None
    To determine the affinity of 3A3 Fab by BLI, anti-human IgG Fc sensors were coated with the anti-foldon antibody identified in this work (3E11) at 20nM in kinetic buffer.
    anti-human IgG
    suggested: None
    anti-foldon
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    MERS (18), HKU1 (18), and the SARS-2 variants HexaPro S2 (residues 697-1208 of the SARS-2 spike with an artificial signal peptide, proline substitutions at positions 817, 892, 899, 942, 986 and 987 and a C-terminal T4 fibritin domain, HRV3C cleavage site, 8xHisTag and TwinStrepTag), HexaPro RBD-locked-down (HexaPro with S383C-D985C substitutions), and aglycosylated HexaPro (HexaPro treated with Endo H overnight at 4 °C leaving only one N-acetylglucosamine attached to N-glycosylation site) as well as MERS S2-only (residues 763-1291 of MERS-2P with 8 additional stabilizing substitutions), MERS S2-apex-less (MERS S2-only construct with residues 811-824 replaced with GGSGGS and residues 1042-1073 replaced with a flexible linker) were expressed in Freestyle 293-F cells (ThermoFisher Scientific).
    293-F
    suggested: RRID:CVCL_6642)
    On day 2 after transfection, HEK-293T-hACE2 cells (BEI, NR-52511), which stably expresses human ACE2, were stained with 1 μM CellTrace Far Red dye (Invitrogen, Ex/Em: 630/661 nm) in PBS for 20 minutes at room temperature, then quenched with DMEM with 10% heat-inactivated FBS for 5 minutes, and resuspended in fresh media.
    HEK-293T-hACE2
    suggested: None
    expressing human ACE2 under an EF1a promoter was used to transduce HEK293T cells.
    HEK293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    Flow cytometry: On day 0, Expi-293 cells (ThermoFisher) were mock-transfected or transfected with pWT-SARS-2-spike (BEI NR-52514) or pD614G-SARS-2-spike (generated by site-directed mutagenesis).
    Expi-293
    suggested: RRID:CVCL_D615)
    Experimental Models: Organisms/Strains
    SentencesResources
    Murine immunization: Three BALB/c mice were immunized subcutaneously with 5μg pre-fusion stabilized MERS S2 and 20 μg of ODN1826 + 100 μl of 2X Sigma Adjuvant System
    BALB/c
    suggested: None
    Software and Algorithms
    SentencesResources
    Images were collected with Zeiss LSM 710 confocal microscope (Carl Zeiss, Inc) and processed using ImageJ software (http://rsbweb.nih.gov/ij) (Fig. 2 and fig.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    The statistical significance of either HEK-ACE2 colocalization percentage or average cell size between different conditions was calculated with ANOVA using GraphPad Prism 7 (GraphPad Software).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Spectra were manually assessed, and figures were prepared using HD-eXplosion (40) and PyMOL (41).
    PyMOL
    suggested: (PyMOL, RRID:SCR_000305)
    Cells were washed again, then scanned for AF647 (640 nm excitation, 670/30 bandpass emission) fluorescence on a BD Fortessa flow cytometer and analyzed with FlowJo (Fig. 6B).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)

    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: We detected the following sentences addressing limitations in the study:
    There are several limitations to this work as currently described. First, a structure showing the atomic details of 3A3 complexed with spike would provide additional insight into the mechanism of binding and neutralization. However, structures of antibodies bound to S2 are generally challenging to obtain with just one structure available of an antibody binding near the HR2 stem (28). It is possible that 3A3 binding distorts spike structure, disturbing otherwise ordered regions. Accordingly, additional efforts to better understanding the molecular underpinnings of 3A3/ spike interactions are underway. Second, while we have shown that 3A3 binds spike from all three highly pathogenic coronaviruses with similar affinities, we have only demonstrated its ability to neutralize SARS-2 spikes in vitro. Demonstration of broad neutralization in addition to broad recognition would increase the potential relevance of this epitope for future therapeutics. The 3A3 epitope is highly conserved, with pairwise comparisons showing between 56% and 100% identity to the SARS-2 epitope for MERS and SARS-1, respectively (fig. S14). Since 3A3 affinity for the least similar MERS spike is comparable to that for the SARS-2 spike and greater than for HKU1, it seems likely that binding and neutralization depend primarily on RBD position epitope accessibility. The most concerning emerging SARS-2 variants have one conservative substitution in this epitope in B.1.1.7, identified in the United Kingdom, and has...

    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 scite Reference Check: We found one citation with an erratum. We recommend checking the erratum to confirm that it does not impact the accuracy of your citation.

    DOIStatusTitle
    10.1371/journal.ppat.1000863Has correctionIn Vitro Reconstitution of SARS-Coronavirus mRNA Cap Methyla…

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