Neutralizing human monoclonal antibodies to poliovirus map to the receptor binding site

Domain: biology
Published: 2026-01-02
Editorial outcome: minor_revision

Abstract
Poliovirus remains a serious threat to human health. Complete eradication of wild-type poliovirus has not yet succeeded, making the development of successful antivirals critical. Microneutralization assays against all three poliovirus serotypes identified a panel of human monoclonal IgGs, which are either serotype-specific or cross-neutralizing. Here, through cryoEM single particle analysis, we solved high resolution structures of four distinct poliovirus-FAb complexes. These antibodies bind to capsids at the circular depression (canyon) surrounding the icosahedral five-fold symmetry axis, which is also the binding site of the poliovirus receptor (PVR). Analysis of these structures confirms overlap of FAb contacts on the viral capsid with those of PVR. For three of the FAbs, the capsid residues are identified that dictate serotype-specific recognition. Contacts for the cross-neutralizing mAb 10D2 are located deep in the capsid canyon. These structural analyses indicate that antibody competition with the receptor likely leads to neutralization of virus particles and inhibition of poliovirus entry into host cells. Thus, the human IgGs studied here may facilitate development of therapeutics for the ongoing efforts in global eradication of poliovirus.

Introduction
Poliovirus (PV) eradication efforts have been implemented for several decades, with the first vaccination programs beginning in the 1950s1,2. Poliovirus route of infection is through pharyngeal and fecal–oral transmission, with most cases presenting as asymptomatic. Gastrointestinal symptoms occur in ~5% of cases, with the severe clinical outcome of acute flaccid paralysis occurring in ~1% of cases3. There are three serotypes of poliovirus, distinguished by differences in genome sequences and conformational epitopes4. Exposure to one serotype of PV does not protect against others serotypes, and current vaccination programs inoculate against all three serotypes of PV. Vaccination efforts have had a profound effect on the eradication of wild-type poliovirus, with serotypes 2 and 3 declared eradicated, though serotype 1 remains endemic to Afghanistan and Pakistan5. Complete eradication of poliovirus has been stymied by vaccine hesitancy and the necessary continued use of the live attenuated vaccine. Although stable and easy to administer, the Sabin attenuated virus in the oral PV vaccine can revert to a virulent state, resulting in vaccine-derived polioviruses (VDPVs)6–8. Circulating VDPVs (cVDPV) of all three serotypes are present globally and have the potential to spread throughout under-immunized communities, including areas where wild-type polio transmission has been effectively eradicated previously8,9. In addition to cVDPV, derivatives of Sabin strains used in OPV can establish chronic infection in individuals with primary immunodeficiency10. Thus, the development of new antivirals is critically necessary to support efforts for the eradication of poliovirus.

Poliovirus is a positive sense ssRNA virus in the family Picornaviridae. The viral capsid is ~30 nm in diameter and comprises four structural proteins (VP1–4) arranged with T=1 (pseudo-T=3) icosahedral symmetry. Structural features of these viruses include a raised plateau termed the mesa at the five-fold symmetry axis, which is surrounded by a depression known as the canyon11. The canyon is the site of poliovirus receptor (PVR) binding12–14. At the bottom of the canyon is an opening to a hydrophobic pocket that contains a lipid molecule, or pocket factor (PF), which stabilizes the capsid. Upon PVR binding, this lipid exits the pocket, allowing conformational changes to occur that result in the formation of the altered particle, or A-Particle. The A-Particle is a true entry intermediate, which is characterized by the externalization of VP4 and the N-termini of VP1 from the interior of the capsid. These conformational changes further facilitate particle entry and genome release within the host cell15–19. Since the receptor-binding site is conserved across all three serotypes of poliovirus and is essential for virus entry, this site might be exploited for the development of biologics.

High-resolution 3D structures of poliovirus, both alone and with receptor or antibody fragments bound, have given insight into receptor contacts and neutralizing epitopes on the capsid surface14,20–24. The neutralizing epitopes of poliovirus were first determined using escape mutant analysis in mice25–27 and cryoEM structures have previously characterized neutralizing epitopes of poliovirus by mapping both human–chimpanzee chimeric antibodies and camelid nanobodies to the PV capsid24,28. Recently, high-resolution cryoEM structures from our group demonstrated that 9H2, a human monoclonal antibody neutralizing all three PV serotypes, had a distinct epitope compared to those of established murine models23.

Patient-derived, human monoclonal IgGs have been previously isolated as potential biologic candidates. Five of these mAbs (9H2, 10D2, 5E12, 2E1, and 6B5) have been shown to neutralize poliovirus in vitro. Both 9H2 and 10D2 are cross-neutralizing, neutralizing more than one PV serotype, whereas the others are serotype-specific23,29. Previous analysis of 9H2 showed that this antibody binds around the five-fold icosahedral symmetry axis of the PV capsid, overlapping the PVR-binding site, outcompetes soluble PVR for capsid binding, and for PV1 causes expulsion of PF. Here, we have solved the high-resolution structures of poliovirus with fragments antigen-binding (FAbs) of the neutralizing mAbs 10D2, 5E12, 2E1, and 6B5. Through structural analyses, we have determined that all four mAbs bind around the five-fold icosahedral symmetry axis similarly to 9H2; no major conformational changes were induced by FAb binding, and pocket factors are retained in each complex. We were able to map each FAb to the capsid to identify conformational epitopes and the footprints. We determined serotype-specific interactions of these antibodies and showed that contacts overlap with both mAb 9H2 and the poliovirus receptor. This investigation adds considerable information to what residues trigger PF release, induce A-particle formation, and dictate serotype-specificity. Thus, these specific results may aid us significantly in advancing the development of biologicals against PV.

Results
Five-fold binding FAbs saturate PV capsid surface
To investigate the conformational epitopes of these antibodies by cryoEM, fragment antigen binding (FAb) was generated by papain digestion and purified. These FAbs were incubated in excess (3 FAb: 1 virus asymmetric unit) with either Sabin inactivated PV1 (5E12), wild-type PV2 (10D2) or Sabin-inactivated PV3 (2E1 and 6B5) for 30 min at room temperature. Resulting complexes were screened by negative stain TEM and vitrified on continuous carbon-coated Quantifoil grids prior to cryoEM data collection. Data were collected using either a Talos Arctica 200 kV microscope at the Penn State cryoEM facility or an FEI Titan Krios G2i microscope at the University of Minnesota Hormel Institute. Micrographs of all complexes showed virions with densities protruding from the capsid surface, consistent with bound Fab and central capsid densities that were consistent with the packaged genome (Fig. S1a).

Reconstruction and model refinement of PV–FAb complexes
For each PV–FAb complex, several rounds of particle picking and 2D classification were used to select the best 2D classes for homogeneous 3D reconstruction with icosahedral symmetry imposed. After reconstruction and refinement, the resolution of the complex maps ranged from ~2.8 to 3.7 Å. For each complex map, the density magnitude of FAb was equal to that of the capsid as seen in the central section (Fig. 1), which also showed amorphous genomic density. The 3D map for each complex showed a similar binding mode with all 60 symmetry-related copies of the FAb bound to the capsid around the five-fold axis without apparent steric hindrance (Fig. 1a). Local resolution mapping of each complex showed poorer resolution at the hinge region of the FAb, likely due to the inherent flexibility of this structure (Fig. S1D). Overall, local resolution showed strong, even resolution throughout the capsid and no regions of poor resolution induced by FAb binding. Thus, the complex maps were of sufficient resolution and quality for modeling both the virus capsid and the FAb variable domain. For the capsid, the build was initiated using the previously published complex structures of FAb 9H2 and poliovirus (PDB ID 8E8Z, 8E8S, and 8E8X for SIPV1, PV2, and SIPV3-binding complexes, respectively)23. Protein models for the FAb variable domains were generated by submitting the sequence to SabPred AbodyBuilder230. The build for each FAb was initiated with the model placed into the variable domain and adjusted throughout all residues by fit. This initial placement enabled building for all three complementarity-determining regions (CDRs) for both the heavy and light chains for all four mAbs, along with the entirety of each framing region. For all complex maps, capsid proteins VP1–VP4 were also built successfully and included residues VP1 22–302, VP2 9–273, VP3 1–238, and VP4 residues 2–15 and 24–68.

No gross conformational changes were observed upon FAb binding
Possible conformational changes imposed by FAb binding to viral capsids were evaluated by superimposing each of our capsid models with the corresponding PV capsid crystal structures (PDB ID 1HXS, 1EAH, and 1PVC for PV1, PV2, and Sabin PV3, respectively)31–33. The ɑ-carbon root mean squared deviation (RMSD) for the FAb–virus complexes ranged from ~0.5 to 1.1 Å compared to their representative crystal structures, constituting a lack of global conformational change across each capsid. Possible conformational changes of the capsid were also investigated by comparing the complexes of Fab-PV capsids presented here and previous virus complexes with cross-neutralizing antibody 9H223. The ɑ-carbon root mean squared deviation (RMSD) for the FAb–virus complexes once again ranged from ~0.5 to 1.1 Å compared to their 9H2-PV counterparts (Table S2). While no gross conformational changes were observed, local conformational changes within the FAb interface are described, ranging from ~3 to 7 Å RMSD per residue compared to representative crystal structures (Fig. S3). Common sites of conformational changes include the VP1 BC loop 95–103 and VP1 GH loop 230–237, which are also common sites of conformational changes induced by poliovirus receptor binding. The GH loop of VP1 is in the doorstop “up” position for each complex compared to their reference crystal structures, a conformation that is present in both receptor-bound and A-particle conformations of PV capsids14,17 (Fig. S4).

Density for the pocket factor molecule, in most cases equal in magnitude to that of the viral capsid, is present in all virus–FAb complexes shown (Fig. S5). This corroborates previous findings of pocket factor density observed in complexes formed with cross-neutralizing antibody 9H2 to both PV2 and PV3 capsids, but not PV123. Interestingly, here strong pocket factor density for the SIPV1–5E12 complex is also observed, equal in density to that of the capsid. These findings, coupled with the presence of genome density and density for the interior capsid protein VP4 within the EM map, confirm that FAb binding does not trigger conformational changes leading to the formation of the Altered-particle entry intermediate.

Capsid contacts for each antibody overlap with PVR and 9H2 binding sites
FAb–virus contacts were defined as amino acid residues on the capsid within 0.4 Vander Waal’s radii of FAb residues34. All FAbs bind near the five-fold icosahedral symmetry axis of the PV capsid, with contacts mapping into and near the canyon region (Fig. 2, Table 1A). Each FAb contacts VP1, with additional contacts mapping to VP2 of the same protomer in all complexes except for the SIPV3–2E1 complex. Contacts on the VP3 chain of the neighboring protomer were seen in all complexes except for the PV2–10D2 complex. These contacts with the virus surface make the footprints of 6B5, 2E1, and 10D2 highly similar. However, the footprint of 5E12 was unique with respect to contacts located on the VP1 chain of the neighboring protomer (Fig. 2).

Each mAb has contacts on the virus capsid that overlap with those of the poliovirus receptor, ranging from 3 to 13 total common contacts per mAb (Figs. 3, S2 and Table 2). Most common contacts map to VP1, with a few contacts on either VP2 or VP3 that overlapped for any complex. Of the four FAbs, only two (6B5 and 5E12) shared overlapping contacts with all three exterior capsid proteins VP1–3 in their footprints, whereas the 2E1 footprint shared overlaps with VP1, VP3, and 10D2, which only shared common contacts with VP1 (Table 2).

Each mAb also has several contacts shared with 9H2, the previously characterized pan-neutralizing mAb that also binds at the five-fold symmetry axis on the PV capsid. The total number of shared contacts between 9H2 and each mAb ranges from 5 to 9 contacts, with most of these on capsid protein VP1. MAb 10D2 has significant overlap with 9H2 compared to the other mAbs shown, with 8 of its 10 total capsid contacts overlapping directly with 9H2’s PV2 footprint (Fig. S7, Table S3).

Regarding the paratope of each mAb on the PV capsid surface, each FAb protein had variable numbers of residues binding to the viral capsid, ranging from 6 to 16 total residues per complex (Table 1B). Most of these contacts were from the FAb heavy chain, with a small portion of contacts (2–7 total) from the light chain. Most contacts were contributed by the complementary-determining regions (CDRs) of the FAb variable domain, namely CDR3 for both the heavy and light chains, with contacts from the FAb-framing regions only present for two of the PV–FAb complexes (Table S4).

Contacts conferring serotype-specific neutralization of five-fold-binding mAbs
Some of the mAbs presented have “sequence-equivalent” contacts, those at the same amino acid position in the capsid proteins when the three serotype sequences are aligned (Fig. S13). The comparison of these contacts indicates which interactions drive serotype-specific binding and neutralization. The mAb 5E12 specifically neutralizes only PV1, whereas 6B5 and 2E1 neutralize only PV3. While there are differences in identity of amino acid residues at some positions, two specific residues at positions 100 and 168 were considered as being critical to serotype-specific neutralization of these mAbs, due to the lack of conservation between each of the three PV serotypes.

5E12 and 2E1 both contact the residue at VP1 position 168. In SPV1, this residue is a glutamate and is predicted to make hydrogen bonds and electrostatic interactions with residue R100 in CDR3 of the 5E12 heavy chain. Conversely, in PV3, this residue is a lysine, leading to electrostatic repulsion and subsequent lack of binding at this residue position. This lack of contact is also true for PV2, where there is a glycine residue that is unable to form a predicted H-bond with R100. Conversely, 2E1 makes electrostatic interactions with VP1 K168 through residue E56 in its light chain, whereas repulsive charge contacts at E168 of SPV1 lead to the lack of contact at this position (Fig. 4A and B).

Another important site is found at position 100 of VP1. Both 5E12 and 6B5 contact this residue, an asparagine in type 1 poliovirus and arginine in both type 2 and type 3 poliovirus. For 5E12, predicted hydrogen bonds between the terminal amine at residue N1 of the heavy chain and the carbonyl in the N100 side chain mediate contact at this residue, whereas the side chain of the arginine residue for PV3 would sterically clash with the n-terminal amine of the heavy chain variable region, preventing binding. In the case of 6B5 binding to residue VP1 R100, the terminal carbonyl groups of D23 in the 6B5 heavy chain make electrostatic contacts with R100, whereas the N100 side chain in SPV1 is out of position to contact D23 (Fig. 4C and D).

Contacts in the canyon floor confer cross-serotype neutralization of poliovirus in mAB 10D2
The 10D2 mAb described here neutralizes serotypes 2 and 3, whereas the 9H2 mAb analyzed previously neutralizes all three serotypes of PV. Antibody 9H2 recognizes capsids via a CDR loop that binds into the canyon, where residue S128 in 9H2’s heavy chain is predicted to make hydrogen bonds with residues D114 and W108, adjacent to the pocket factor molecule23. The CDR loop 3 of mAb 10D2 also binds into the canyon floor, with residue G110 contacting residue D114 of VP1. Additionally, the R-group terminal alcohol in residue T108 of the 10D2 CDR3 makes a predicted hydrogen bond with V107’s carboxyl group and overlaps with residue W108 on VP1. Each of these contacts, along with contacts in the VP1 GH loop at residues 223 and 226, helps stabilize the CDR loop 3 in the canyon floor (Fig. 5).

Five-fold binding mAbs neutralize as FAb fragments
The distance of the heavy chain C-termini of each FAb ranges from 59 to 75 Å, with symmetry-related FAb copies angled away from one another, tangential to the capsid. This binding mode, coupled with the binding angles relative to the capsid, would prevent bivalent binding and accommodate monovalent binding to the capsid35,36. The mode of binding is similar to the monovalent binding of 9H2 FAb to PV capsids, which was sufficient to neutralize serotypes 1 and 3 of poliovirus in vitro23.

Neutralization efficacy of each of the mAbs presented here towards either SPV1 or SPV3 was assessed by microneutralization assays with the full-length mAb, with effective neutralization concentrations ranging from 2.0*10−2 to 2.0*10−6 mg/mL. We performed further microneutralization assays with 10-fold serial dilutions of FAb fragments towards either SPV1 (for 5E12) and SPV3 (for all other mAbs). All four antibodies neutralized the virus as FAb, with effective neutralizing concentrations ranging from 2.0*10−2 to 2.0*10−5 mg/mL (Fig. S14).

Methods
Mammalian cell culture and viruses
HeLa or Vero cells (ATCC) were infected with poliovirus inoculum at an MOI of 0.1–4 and propagated until >90% CPE was achieved. The virus was then titered by plaque assay as described previously23. Sabin serotypes 1 and 3 were kindly provided by Konstantin M. Chumakov, previously at CBER-FDA. After data collection, all wild-type PV2 inoculum, stocks, and associated materials were destroyed in accordance with the US CDC, according to their exact instructions. Sabin-inactivated poliovirus was supplied by the Beijing Institutes of Biological Sciences, China. Virus stock was exchanged against PV buffer (50 mM HEPES, pH 8, 150 mM NaCl, 3 mM MgCl2), as described previously23.

Virus propagation and purification
Poliovirus strains were propagated and purified as previously described23. Each virus propagation for purification was conducted in a 10 and 2-stack cell factory (VWR). After infection, the cell lysate was frozen and thawed 3 times and centrifuged at 15,000 × g for 10 min to pellet cell debris. The debris was resuspended and Dounce homogenized with the addition of 10% NP40 (Thermo Fisher). The homogenization was combined with the supernatant and centrifuged at 15,000 × g for 10 min. The resulting supernatant was transferred to a 4 L beaker, PEG 8000 was added to 5%, NaCl was added to a final concentration of 0.5 M, and stirred overnight at 4°C. The mixture was centrifuged at 15,000 × g for 10 min to pellet the PEG-aggregated virus, which was resuspended in a minimal amount of PV Buffer (50 mM HEPES, pH 8, 200 mM NaCl, and 3 mM MgCl2 buffer). 0.5 M MgCl2 was added to 10% of the sample volume, followed by DNAse I (Spectrum) and SDS 10–5% of the sample volume, and incubated at RT for 30 min. Per mL of sample, 0.8 mg trypsin and 0.15 mL of 0.5 M EDTA, pH 9.5, were added and incubated at 37°C 10 min. N-Lauryl Sarcosine 10% was added to 10% of the sample volume. This sample was then transferred to 50.2 Ti Beckman tubes and 2 mL 30% sucrose in the sample buffer layered beneath it. Tubes were transferred to the Beckman 50.2 Ti rotor for ultracentrifugation at 277,688 × g for 2 h at 4°C with slow braking. The supernatant was discarded, and the viral pellet rehydrated in 1 mL sample buffer at 4°C overnight. The fully resuspended virus was applied to a continuous 10–40% K-tartrate gradient, balanced in the SW41 rotor, and ultracentrifuged at 222,000 × g for 1.5 h at 4°C with no brake. Resulting virus bands were collected into a syringe by piercing the ultracentrifuge tube with an 18 G needle. Virus was concentrated, and the buffer was exchanged by ultracentrifugation at 277,688 × g and resuspension in PV Buffer. Concentration was estimated by spectrophotometry (DeNovix).

FAb generation from five-fold binding mAbs
All mAbs were provided by the PATH Center for Vaccine Access and Design. Antigen-binding fragments (FAb) were digested using Pierce FAb Micro Preparation Kits (Thermo Fisher, cat# 44685) and purified using Protein A microcentrifuge columns. Purified FAb was further concentrated using tabletop microcentrifuge columns (Millipore-Sigma).

Microneutralization assays
Microneutralization assays for five-fold-binding mAbs and FAbs were performed as previously described, with minor modifications23. Vero cells grew to ~80% confluence in DMEM, 1% NEAA, and 10% FBS media in 96-well plates. 50 µL 1400 pfu/mL SPV1 or SPV3 virus inoculum was incubated with 50 µL of mAb or FAb (ranging from 2.0*10−2 to 2.0*10−9 mg/mL) in DMEM, 1% NEAA for 1 h at 32°C prior to application to PBS-washed cells. After incubation at 32°C for 1 h, 50 µL DMEM, 1% NEAA, 5% FBS media was added to each well. Plates were incubated at 32°C and monitored for CPE by microscopy for 72 h.

Sample preparation and cryoEM data collection
Poliovirus-FAb complexes were made by incubating FAb and PV together at a ratio of 3 FAb: 1 binding site for 30 min at room temperature. 3.5 µL of complex was applied to glow-discharged 2/1 Cu Quantifoil grids with 3–4 nm continuous carbon coating. Samples were incubated for one minute, blotted, and vitrified using an MK III Vitrobot (Thermo Fisher) operating at 4°C and 100% humidity. CryoEM datasets for 5E12 and 10D2-PV complexes were collected at 200 kV with a Talos Arctica cryo-TEM (Thermo Fisher) at the Penn State CryoEM facility, and datasets for 2E1 and 6B5-PV complexes were collected using a 300 kV Krios G2i cryo-TEM (Thermo Fisher) at the Hormel Institute. EPU software was used for automated data collection with defocus ranges from −0.5 to −2 µm for each PV-FAb complex. The Talos Arctica was equipped with a Falcon IV direct electron detector at a magnification of ×120,000, yielding a pixel size of 1.3 Å. The Krios G2i was equipped with a Gatan K3 direct electron detector operating at ×59,000 magnification, yielding a pixel size of 1.1 Å (Table S1).

cryoEM image processing
All cryoEM image processing was performed in CryoSparc46. Micrographs were motion corrected, and patch CTF estimation was performed using the “Patch CTF” job. Particles were picked using the “blob picker” and “template picker” jobs, extracted from micrographs, and selected for those with discernible density for the FAb fragment on the capsid and densities corresponding to the intact genome. The best particles were then selected for homogeneous refinement, using an initial model for each complex generated through ab-initio reconstruction and with imposed I1 symmetry (Figs. S9–S12).

Model building and contact identification
Preliminary models for FAb were generated by submitting the variable domain sequences to SabPred AbodyBuilder2. Initial build of the viral capsid was performed using PDB models 8E8Z, 8E8S, and 8E8X for SIPV1, PV2, and SIPV3-binding mAbs, respectively23. Model building and refinement were performed using the ISOLDE plug-in in UCSF ChimeraX47,48 and Phenix49, before model validation with MolProbity50. Map-to-model fit was assessed using the Q-Score plugin of ChimeraX51. FAb contacts on the virus surface were examined in UCSF Chimera34 and defined as residues having atoms separated by <0.4 Å van der Waals radius. Palmitate was modeled as the pocket factor, as it is the most common pocket factor for enteroviruses52. Roadmaps defining contacts for FAb, 9H2, and PVR contacts on the virus surface were generated using RIVEM53. Figures were generated using ChimeraX. Sequence alignments between PV strains were performed using Clustal Omega54 and imaged with JalView55.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Poliovirus eradication is increasingly uncertain, spurring important research in the field. Building upon their recent publication (Nat Comm 2023), the authors used cryo-EM to solve high resolution structures of four poliovirus-Fab complexes. The Fabs, obtained from neutralizing mAbs 10D2, 5E12, 2E1 and 6B5, bound to icosahedral poliovirus particles in a canyon around the 5-fold axis of symmetry. Contacts with serotype-specific capsid residues were identified for three of the Fabs while a cross-neutralizing mAb (10D2) contacts conserved residues located deep in the capsid canyon, reinforcing an outcome reported before (Nat Comm 2023). The Fabs bind to overlapping regions of the canyon required for binding of the poliovirus receptor, suggesting a mechanism of neutralization that prevents receptor binding and entry. Intriguingly, unlike binding of the poliovirus receptor, the Fabs do not provoke large conformational changes in capsid structures nor induce release of pocket factor. Altogether, this study highlights how Fab-poliovirus protein-protein interactions that neutralize poliovirus particles are similar, yet distinct from receptor-poliovirus protein-protein interactions that trigger various aspects of virus binding and entry (pocket factor release, A-particle formation and genome release).\nThe authors conclude that serotype-specific mAbs contact serotype-specific residues of capsid proteins whereas cross-neutralizing mAbs contact more conserved residues deep in the capsid canyon.\nCritique: The current investigation is consistent with, and builds upon, a large body of work in the field. The data for 10D2 cross-neutralizing antibodies reported herein reinforce those previously reported for 9H2 (Nat Comm 2023): cross-neutralizing antibodies contact conserved residues deep in the capsid canyon. Altogether, this study highlights and compares Fab-poliovirus protein-protein interactions that neutralize poliovirus particles with receptor-poliovirus protein-protein interactions that trigger various aspects of virus binding and entry (pocket factor release, A-particle formation and genome release). Unlike binding of the poliovirus receptor, the Fabs do not provoke large conformational changes in capsid structures nor induce release of pocket factor.\nThe data are of high quality, supporting the authors important conclusions.\nI have only minor points for the authors’ consideration.\nMinor points\n1. Line 102. Awkward phrase...biologicals towards poliovirus.\n2. Line 164. Spell pocket factor.\n3. Discussion, lines 262-273. Is there a logical reason why distinct poliovirus epitopes are reported for murine and human antibodies?", "reviewer_2": "The main new findings are the role of residue 100 in the restriction of some antibodies to specific serotypes, and a proposed role of residue 234 in pocket factor release. The structural work is new but does not bring the type of conceptual or mechanistic insights expected in Nature Communications. This is all the more important since the “real-life” impact of this work is not directly evident. A useful antibody exists (9H2) and there is no real evidence that the delivery of antibodies is a viable solution in the countries affected by endemic poliovirus 1 or outbreaks of vaccine-derived poliovirus outbreaks. These are often remote locations, war zone or under-resourced regions where injection with purified antibody is not an option.\nFundamentally, the mechanism of antibody-induced release of the pocket factor would be interesting, but this study does not provide data beyond static structures (indeed, these antibodies don’t induce the release of the pocket factor).\nAs for the form, the design and quality of the figures does not support the interpretations. They are difficult to read at best and do not always illustrate the point made in the text:\nFig. 1 belongs to the supplementary material (at least for panels B and C).\nFig. 2 is very hard to read. The black colour and choice of shadows/rendering makes it uninterpretable. Zoomed insets on what is to take way from the figure would help.\nFig. 4: again, poor rendering choice. White-on-white hydrogen atoms are almost invisible.\nFig. 6: Is it not evident at all from Fig. 6 that residue 234 is \"critical for pocket factor release\". We just see a couple of loops that swing in/out but don't have any idea of pocket size, opening, surface properties.\nTechnically, I’m surprised that datasets with 10 times more particles and a better microscope/detector setup yields slightly worse resolution to the Glacios one (~2.9Å). They should be much better (and the local resolution map support that the global resolution is better than stated). Whether it is an error in reporting or in data processing, this is concerning for a straightforward Cryosparc workflow.\nDetailed comments:\nl. 23-24. “vaccine-derived poliovirus (VDPV) has exacerbated the current situation ”: In the current anti-vaccine climate, I would be cautious with the way these points are articulated.\nl. 35-36: How are these going to be delivered in practice in area of concern? Is serotype-specificity important?\nl. 44, \"most severe clinical outcome of acute flaccid paralysis\": no, the most severe clinical outcome is death.\nl. 82, If this one antibody (9H2) is known and broad-spectrum, is there still a need for new mAbs?\nl. 244, monovalent binding: If it's true that the binding of intact IgG is monovalent, this should be measurable in neutralisation assays with similar EC50s between Fab and IgG when corrected for concentration. Is this the case? The figure supporting the monovalent binding, tangential orientation etc. should be presented in supplementary material."}, "author_response": "We appreciate the comments by Reviewer 1.\nI have only minor points for the authors’ consideration.\nMinor points\n1. Line 102. Awkward phrase...biologicals towards poliovirus.\nThe text was changed to be less awkward.\n2. Line 164. Spell pocket factor.\nThis has been corrected.\n3. Discussion, lines 262-273. Is there a logical reason why distinct poliovirus epitopes are reported for murine and human antibodies?\nCitations have been added to this passage. Historically, epitope mapping of poliovirus was done with murine antibodies and escape mutants (25-27). Chumakov (24) reported the first epitope mapped with a chimpanzee-human chimeric antibody, which mapped to a different location on the capsid from any of the work done with murine antibodies. Our lab reported the first epitope mapped to the virus capsid with a human monoclonal, 9H2 (23). Here we report how four more human monoclonal antibodies recognize the capsid. We have been surprised throughout these studies that there is little to no overlap between human and murine antibodies on the capsid.\nReviewer #2 (Remarks to the Author):\nThe main new findings are the role of residue 100 in the restriction of some antibodies to specific serotypes, and a proposed role of residue 234 in pocket factor release. The structural work is new but does not bring the type of conceptual or mechanistic insights expected in Nature Communications. This is all the more important since the “real-life” impact of this work is not directly evident. A useful antibody exists (9H2) and there is no real evidence that the delivery of antibodies is a viable solution in the countries affected by endemic poliovirus 1 or outbreaks of vaccine-derived poliovirus outbreaks. These are often remote locations, war zone or under-resourced regions where injection with purified antibody is not an option.\nFundamentally, the mechanism of antibody-induced release of the pocket factor would be interesting, but this study does not provide data beyond static structures (indeed, these antibodies don’t induce the release of the pocket factor).\nAs for the form, the design and quality of the figures does not support the interpretations. They are difficult to read at best and do not always illustrate the point made in the text:\nFig. 1 belongs to the supplementary material (at least for panels B and C).\nPanels C showing fit to map have been moved to Supplemental leaving only panels A and B showing surface-rendered and central sections of maps. For complexes, illustrating the central section is necessary to show the quality of the map and the density magnitude of the bound protein relative to that of the capsid.\nFig. 2 is very hard to read. The black colour and choice of shadows/rendering makes it uninterpretable. Zoomed insets on what is to take way from the figure would help.\nThe figure has been reworked with changes in color and the addition of zoomed insets to aid interpretation.\nFig. 4: again, poor rendering choice. White-on-white hydrogen atoms are almost invisible.\nThe figure has been reworked with changes in color to aid interpretation.\nFig. 6: Is it not evident at all from Fig. 6 that residue 234 is \"critical for pocket factor release\". We just see a couple of loops that swing in/out but don't have any idea of pocket size, opening, surface properties.\nTo address the Reviewer’s concern, we have reworked the figure, edited and added text, and included additional analysis. These changes add clarification to the presentation of residues critical for pocket factor release.\nTechnically, I’m surprised that datasets with 10 times more particles and a better microscope/detector setup yields slightly worse resolution to the Glacios one (~2.9Å). They should be much better (and the local resolution map support that the global resolution is better than stated). Whether it is an error in reporting or in data processing, this is concerning for a straightforward Cryosparc workflow.\nWe have added a description of the maps reporting the resolution as a range, which as the Reviewer points out, is an important description for a reconstruction of a complex where the virus and the site of interaction are often a better resolution than the apical tip of the bound protein.\nWorkflow figures have been added to supplemental.\nTwo data sets (5E12 and 10D2) had about 20,000 particles each collected on the Glacios and attained resolutions of 2.84 and 3.65, respectively.\nTwo data sets (2E1 and 6B5) consisted of 237k and 450k particles each were collected on the Krios G2 and attained resolutions of 2.95 and 2.94, respectively (Table S1). Both microscopes in question have been benchmarked as capable of producing maps at 1.9 Å resolution. It is not commonly recognized that a 200 kV Glacios or Arctica is capable of 1.9 Å resolution.\nAlthough there is a correlation between particle number and resolution, there are multiple other factors controlling final resolution such as ice thickness and protein flexibility. For both of the data sets collected on the Krios, we noticed areas of thicker ice.\nDetailed comments:\nl. 23-24. “vaccine-derived poliovirus (VDPV) has exacerbated the current situation ”: In the current anti-vaccine climate, I would be cautious with the way these points are articulated.\nWe agree with this caution and have edited the text. The passage in Abstract was deleted entirely.\nl. 35-36: How are these going to be delivered in practice in area of concern? Is serotype-specificity important?\nThere are currently no approved and licensed antivirals or therapeutics such as monoclonals for treatment of iVDPV subjects globally, the treatment of these subjects will be coordinated with WHO GPEI (Global Polio Eradication Initiative) to treat the handful of iVDPV subjects who are chronic poliovirus shedders and specificity of the antibody in this treatment option to such subjects will help to achieve the two main goals of polio eradication (wildtype virus eradication and VDPVs including iVDPV eradication) (10).\nl. 44, \"most severe clinical outcome of acute flaccid paralysis\": no, the most severe clinical outcome is death.\nThe text has been corrected.\nl. 82, If this one antibody (9H2) is known and broad-spectrum, is there still a need for new mAbs?\nA critical challenge to achieving complete eradication of poliovirus circulation will be shedding of live vaccine-derived poliovirus strains by individuals with primary immunodeficiencies (PID). Because live poliovirus shedding can persist for years in these individuals, chronic excretion presents a long-term risk of seeding ongoing VDPV circulation and potentially disease outbreaks (10). A therapeutic intervention which can clear poliovirus infection from a substantial fraction of this population could mitigate this risk. Intervention options including use of individual broad spectrum or in combination with type specific antibodies will be very helpful options.\nl. 244, monovalent binding: If it's true that the binding of intact IgG is monovalent, this should be measurable in neutralisation assays with similar EC50s between Fab and IgG when corrected for concentration. Is this the case? The figure supporting the monovalent binding, tangential orientation etc. should be presented in supplementary material.\nThe measurements to assess whether bivalent binding is possible were established in seminal papers by Elizabeth Hewat and Dieter Blaas (35,36), which are cited in the manuscript. In the four complex structures solved and presented here, none of the Fabs are bound with a geometry consistent with bivalent binding. Specifically (lines 243-246), Fabs are not bound in an orientation relative to the capsid or to the symmetry related neighboring fab that would allow bivalent binding of a Mab. The text has been edited to clarify, with care taken to avoid overstating the analysis."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Waddey and colleagues report that neutralizing human monoclonal antibodies to poliovirus map to the receptor binding site. The overlapping footprints and specific protein-protein interactions of both mAbs and poliovirus receptor are clearly highlighted in the revised Figures and Tables. The cryo-EM structures of poliovirus-FAb complexes are of sufficient resolution to support the author’s important conclusions.\nAs summarized in their response to the previous critiques, the manuscript was revised to improve some aspects of data presentation and to clarify cryo-EM data collection and processing methods. The authors clarified the factors that impact the resolution of poliovirus-FAb complexes. Altogether, revisions in the manuscript were responsive to the previous critiques, improving the clarity and impact of the study.\nTheoretically appealing conceptual frameworks. 1) FAb-poliovirus protein-protein interactions that neutralize poliovirus particles are similar, yet distinct from receptor-poliovirus protein-protein interactions that trigger various aspects of virus binding and entry (pocket factor release, A-particle formation and genome release). Intriguingly, unlike binding of the poliovirus receptor, the FAbs do not provoke large conformational changes in capsid structures nor induce release of pocket factor. 2) Serotype-specific mAbs contact serotype-specific residues of capsid proteins whereas cross-neutralizing mAbs contact more conserved residues deep in the capsid canyon. These are theoretically appealing conceptual frameworks to inspire ongoing work in the field.", "reviewer_2": "The authors have not rebutted the main points from my previous review (1st two paragraphs) and focused on my comments on the figures. From my reading, both reviewers agree that given the previous publication (Nature Communications, 2023) and the absence of a novel mechanism, the work does not bring the type of conceptual advances expected for publication in Nature Communications. Nothing in the revised submission counters these initial views.\nMy concern about the quality of the structural work remains. With 237k and 450k icosahedral particles, the resolution limits should be better. More advanced processing should have been explored."}, "author_response": ""}]

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