Conformational and glycan heterogeneity associated with furin cleavage of Spike as a cause of incomplete neutralization of SARS-CoV-2

Conformational and glycan heterogeneity associated with furin cleavage of Spike as a cause of incomplete neutralization of SARS-CoV-2 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Conformational and glycan heterogeneity associated with furin cleavage of Spike as a cause of incomplete neutralization of SARS-CoV-2 Rajesh Ringe, Sahil Kumar, Rathina Delipan, Chanchal Sharma, Jyoti Jadoun, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6134096/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Nov, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract SARS-CoV-2 Spike is the sole target of neutralizing antibodies (nAbs) but highly resilient to the immune pressure driving continuous genetic evolution. While potency and breadth of neutralization are widely studied, the incomplete neutralization - the mechanism of resistance without needing genetic change, remains unexplored. Several monoclonal antibodies, although potent, showed incomplete neutralization of genetically homogeneous pseudovirus suggesting the existence of distinct spike conformations peculiarly resistant to antibody binding. Incomplete neutralization is more commonly seen with NTD-specific antibodies typically exhibiting shallow neutralization curves in which neutralization saturates well below 100%. The residual infectivity in neutralization is thus accounted for the un-neutralized virions due to their intrinsic resistance to the tested antibody. Although the published studies on spike glycosylation, structure, and conformations provide insights on the spike heterogeneity the precise mechanism for the incomplete neutralization has not been established. In this study, we devised a method to separate the un-neutralized virion population, called as persistent fraction of infectivity (PF), and characterized its on-virus spike protein. The neutralization resistance of PF was stable and was unrelated to the conformational equilibrium that exists in the pseudovirus stock. The spike on the PF was highly cleaved between S1 and S2 subunits, adopted the closed conformation, and expressed more mannosidic glycans on RBD than the total virus population. Our study provides a precise explanation for the incomplete neutralization by the potent neutralizing antibodies and delineate the association between furin cleavage of spike and its conformation and glycosylation. Biological sciences/Microbiology/Virology/Viral immune evasion Biological sciences/Microbiology/Virology/Viral pathogenesis SARS-CoV-2 Spike conformation glycosylation neutralizing antibodies persistent fraction Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SIFiguresandlegends.pdf Figure S1, Figure S2, Figure S3, Figure S4, Figure S5 Cite Share Download PDF Status: Published Journal Publication published 19 Nov, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6134096","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":425457638,"identity":"2d5f1088-0daf-4437-a705-764582c9f6f2","order_by":0,"name":"Rajesh Ringe","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA70lEQVRIiWNgGAWjYHCCBAbGBgYeIIPxAZDg4SNFC7MBSAsbUfYAtYAAmwSYJKTavP3Aw4c/d9yTMedffKzya46dDBsD88NHN/BokTmTkGzMe6aYx3LGs7TbstuSgQ5jMzbOwaNFgiEhTZqxLYHH4MYZs9uS25iBWnjYpPFq4X+Q/vMnWMv5b8WS2+qJ0CKRkMbAC9JyvoeN8eO2w8RoeZAsDdZyg81YmnHbcR42ZkJ+4c9J/Ah0mL3B+cMPP/7cVm3Pz9788DE+LcC4S4BqTmBgBkUoAzNe5SDAfgBC8x9gYPxBUPUoGAWjYBSMRAAAqhJFl/PPATcAAAAASUVORK5CYII=","orcid":"","institution":"CSIR-Institute of Microbial Technology, National Institute of Pharmaceutical Education and Research","correspondingAuthor":true,"prefix":"","firstName":"Rajesh","middleName":"","lastName":"Ringe","suffix":""},{"id":425457639,"identity":"58aaa8cc-6c21-4bbd-80e3-a325f3d492c3","order_by":1,"name":"Sahil Kumar","email":"","orcid":"","institution":"Institute of Microbial Technology, Council of Scientific and Industrial Research (CSIR-IMTECH)","correspondingAuthor":false,"prefix":"","firstName":"Sahil","middleName":"","lastName":"Kumar","suffix":""},{"id":425457640,"identity":"9bd59e38-5b33-4f14-b936-cb05934cfa86","order_by":2,"name":"Rathina Delipan","email":"","orcid":"","institution":"CSIR-Institute of Microbial Technology","correspondingAuthor":false,"prefix":"","firstName":"Rathina","middleName":"","lastName":"Delipan","suffix":""},{"id":425457641,"identity":"ba504927-3c9d-463e-82fd-78b309b10695","order_by":3,"name":"Chanchal Sharma","email":"","orcid":"","institution":"CSIR-Institute of Microbial Technology","correspondingAuthor":false,"prefix":"","firstName":"Chanchal","middleName":"","lastName":"Sharma","suffix":""},{"id":425457642,"identity":"2b5bb3c6-8b50-4cc0-b2d3-a3ddd01e1a48","order_by":4,"name":"Jyoti Jadoun","email":"","orcid":"","institution":"CSIR-Institute of Microbial Technology","correspondingAuthor":false,"prefix":"","firstName":"Jyoti","middleName":"","lastName":"Jadoun","suffix":""},{"id":425457643,"identity":"366d3283-e60d-45bd-aa0b-afd8d341f8dc","order_by":5,"name":"Kawkab Kanjo","email":"","orcid":"","institution":"Indian Institute of Science","correspondingAuthor":false,"prefix":"","firstName":"Kawkab","middleName":"","lastName":"Kanjo","suffix":""},{"id":425457644,"identity":"52149fc0-e38a-44db-ae95-3dfc430f2678","order_by":6,"name":"Randhir Singh","email":"","orcid":"","institution":"Mynvax Pvt. Ltd","correspondingAuthor":false,"prefix":"","firstName":"Randhir","middleName":"","lastName":"Singh","suffix":""},{"id":425457645,"identity":"42af6f87-4d20-4966-b454-3386d9511a0b","order_by":7,"name":"Raju Rajmani","email":"","orcid":"","institution":"Indian Institute of Science Bangalore","correspondingAuthor":false,"prefix":"","firstName":"Raju","middleName":"","lastName":"Rajmani","suffix":""},{"id":425457646,"identity":"676efd41-da1a-4274-be83-a306fb076e15","order_by":8,"name":"Suprit Deshpande","email":"","orcid":"","institution":"BRIC-Translational Health Science \u0026 Technology Institute","correspondingAuthor":false,"prefix":"","firstName":"Suprit","middleName":"","lastName":"Deshpande","suffix":""},{"id":425457647,"identity":"c8ca4452-9e01-4062-aea3-73d8f6888053","order_by":9,"name":"Rajesh Pandey","email":"","orcid":"","institution":"CSIR-Institute of Genomics and Integrative Biology","correspondingAuthor":false,"prefix":"","firstName":"Rajesh","middleName":"","lastName":"Pandey","suffix":""},{"id":425457648,"identity":"31f80d6f-41f3-4edd-8cd9-095fa140869b","order_by":10,"name":"Krishan Thakur","email":"","orcid":"https://orcid.org/0000-0003-4500-2133","institution":"Institute of Microbial Technology","correspondingAuthor":false,"prefix":"","firstName":"Krishan","middleName":"","lastName":"Thakur","suffix":""},{"id":425457649,"identity":"5aa4352d-36b2-4e05-85b9-6bcc78786c1a","order_by":11,"name":"Jayanta Bhattacharya","email":"","orcid":"","institution":"Translational Health Science and Technology Institute","correspondingAuthor":false,"prefix":"","firstName":"Jayanta","middleName":"","lastName":"Bhattacharya","suffix":""},{"id":425457650,"identity":"3f1edd78-8eb0-4401-b911-01874e0be567","order_by":12,"name":"Rogier Sanders","email":"","orcid":"https://orcid.org/0000-0002-2324-8573","institution":"Amsterdam UMC, University of Amsterdam","correspondingAuthor":false,"prefix":"","firstName":"Rogier","middleName":"","lastName":"Sanders","suffix":""},{"id":425457651,"identity":"4d14fcf2-7f6c-4152-a2fd-b9058fc367ad","order_by":13,"name":"Marit J. van Gils","email":"","orcid":"https://orcid.org/0000-0003-3422-8161","institution":"Amsterdam UMC","correspondingAuthor":false,"prefix":"","firstName":"Marit","middleName":"J. van","lastName":"Gils","suffix":""},{"id":425457652,"identity":"18e34068-b5ff-416b-8be3-bef52b467316","order_by":14,"name":"Raghavan Varadarajan","email":"","orcid":"https://orcid.org/0000-0002-0823-7577","institution":"Indian Institute of Science Bangalore","correspondingAuthor":false,"prefix":"","firstName":"Raghavan","middleName":"","lastName":"Varadarajan","suffix":""}],"badges":[],"createdAt":"2025-03-01 10:25:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6134096/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6134096/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-65099-y","type":"published","date":"2025-11-19T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":96354840,"identity":"2ce70f64-5326-474b-ae17-7c6a6738092e","added_by":"auto","created_at":"2025-11-20 08:11:28","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3779164,"visible":true,"origin":"","legend":"","description":"","filename":"PFofSARSCoV2MS01032025.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6134096/v1_covered_de811ecd-c549-4d5f-9374-fc97baf5e220.pdf"},{"id":78109471,"identity":"cb9c8751-6b09-4a00-914b-8b88de41f534","added_by":"auto","created_at":"2025-03-10 04:43:00","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":937944,"visible":true,"origin":"","legend":"Figure S1, Figure S2, Figure S3, Figure S4, Figure S5","description":"","filename":"SIFiguresandlegends.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6134096/v1/dd729a21ecbee8f39f49991d.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Conformational and glycan heterogeneity associated with furin cleavage of Spike as a cause of incomplete neutralization of SARS-CoV-2","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"SARS-CoV-2, Spike conformation, glycosylation, neutralizing antibodies, persistent fraction","lastPublishedDoi":"10.21203/rs.3.rs-6134096/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6134096/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"SARS-CoV-2 Spike is the sole target of neutralizing antibodies (nAbs) but highly resilient to the immune pressure driving continuous genetic evolution. While potency and breadth of neutralization are widely studied, the incomplete neutralization - the mechanism of resistance without needing genetic change, remains unexplored. Several monoclonal antibodies, although potent, showed incomplete neutralization of genetically homogeneous pseudovirus suggesting the existence of distinct spike conformations peculiarly resistant to antibody binding. Incomplete neutralization is more commonly seen with NTD-specific antibodies typically exhibiting shallow neutralization curves in which neutralization saturates well below 100%. The residual infectivity in neutralization is thus accounted for the un-neutralized virions due to their intrinsic resistance to the tested antibody. Although the published studies on spike glycosylation, structure, and conformations provide insights on the spike heterogeneity the precise mechanism for the incomplete neutralization has not been established. In this study, we devised a method to separate the un-neutralized virion population, called as persistent fraction of infectivity (PF), and characterized its on-virus spike protein. The neutralization resistance of PF was stable and was unrelated to the conformational equilibrium that exists in the pseudovirus stock. The spike on the PF was highly cleaved between S1 and S2 subunits, adopted the closed conformation, and expressed more mannosidic glycans on RBD than the total virus population. Our study provides a precise explanation for the incomplete neutralization by the potent neutralizing antibodies and delineate the association between furin cleavage of spike and its conformation and glycosylation.","manuscriptTitle":"Conformational and glycan heterogeneity associated with furin cleavage of Spike as a cause of incomplete neutralization of SARS-CoV-2","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-10 04:42:53","doi":"10.21203/rs.3.rs-6134096/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"cd222029-f072-441a-8f88-e6ebb4bbaa32","owner":[],"postedDate":"March 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":45345190,"name":"Biological sciences/Microbiology/Virology/Viral immune evasion"},{"id":45345191,"name":"Biological sciences/Microbiology/Virology/Viral pathogenesis"}],"tags":[],"updatedAt":"2025-11-20T08:11:20+00:00","versionOfRecord":{"articleIdentity":"rs-6134096","link":"https://doi.org/10.1038/s41467-025-65099-y","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-11-19 05:00:00","publishedOnDateReadable":"November 19th, 2025"},"versionCreatedAt":"2025-03-10 04:42:53","video":"","vorDoi":"10.1038/s41467-025-65099-y","vorDoiUrl":"https://doi.org/10.1038/s41467-025-65099-y","workflowStages":[]},"version":"v1","identity":"rs-6134096","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6134096","identity":"rs-6134096","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}