Persistent SARS-CoV-2 Spike is Associated with Localized Immune Dysregulation in Long COVID Gut Biopsies

Persistent SARS-CoV-2 Spike is Associated with Localized Immune Dysregulation in Long COVID Gut Biopsies | 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 Persistent SARS-CoV-2 Spike is Associated with Localized Immune Dysregulation in Long COVID Gut Biopsies Marcelo Freire, Salim Abraham Soria, Patrick Peterson, Michael Tankelevich, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9284759/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract SARS-CoV-2 persistence is a proposed driver of Long COVID (LC), but the in-situ relationship between residual viral antigen and immune dysregulation remains poorly defined. To address this critical gap, we employed a high-resolution, multi-modal approach—combining RNAscope, GeoMx Digital Spatial Profiling (DSP), spatial transcriptomics, and multiplex immunofluorescence—on 25 terminal ileum and left colon biopsies from a clinical cohort of 8 LC participants and 5 healthy controls. We confirmed the persistence of SARS-CoV-2 Spike transcript and protein in the gut tissue of all LC cases and controls tested. Yet, comparison of Spike-positive (Spike+) regions in LC versus healthy control colon tissues revealed a differential, symptomatic state-associated signature, with 57 differentially expressed genes (DEGs) (26 upregulated, 31 downregulated), revealing genes that disrupt the immune response in LC subjects. LC colon Spike+ regions demonstrated increased expression of AQP8 and other absorptive-related genes (SLC26A3, SLC26A2, and CLCA4) which are involved with Chron’s disease along with transcripts involved in tumorigenesis (GUCA2A, S100P, TSPAN1). Simultaneous downregulation of key homeostatic chemokines (CXCL13, CCL19, CCL21), and other transcripts reported to exhibit low expression in colorectal cancers (TMEM88B, NIBAN3, DMBT1), suggesting a paradox of epithelial tissue stress yet dysfunctional immune trafficking. Further analysis comparing Spike+ versus Spike- regions within LC colon tissue demonstrated an active, localized, antigen-driven immune microenvironment, identifying 122 DEGs (82 upregulated, 40 downregulated), including tumorigenesis genes. Cellular deconvolution of Spike+ regions revealed a statistically significant focal enrichment of myeloid-derived cells (macrophages, non-classical/intermediate monocytes), plasma cells, and regulatory T cells, coupled with significant enrichment in T-cell-related pathways, including ″Antigen processing and presentation,″ and ″Th1/Th2/Th17 cell differentiation″. The ileum displayed a similar, though less pronounced, signature, demonstrating these statistically significant findings are specific to the colon of LC subjects. In contrast, corresponding Spike+ vs. Spike- analysis in healthy control colon tissues showed a more modest transcriptional response with 38 DEGs. Our data provide robust evidence that persistent SARS-CoV-2 Spike protein detection in the gut is not immunologically inert. Instead, it is actively associated with distinct, immune cell composition shifts and a dysfunctional pro-inflammatory transcriptional profile, supporting the hypothesis that retained viral antigen drives chronic immune dysregulation in tissue of Long COVID subjects. Biological sciences/Immunology/Infectious diseases Biological sciences/Immunology/Inflammation Long COVID Gut Biopsies Viral Persistence Spatial Transcriptomics Immune Dysregulation Full Text Additional Declarations There is NO Competing Interest. Supplementary Files GutSupplementaryNatureComms.pdf Supplementary Figures Cite Share Download PDF Status: Under Review 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-9284759","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":619832558,"identity":"50e07d33-2bfb-459b-acdd-cb6f080c7ab5","order_by":0,"name":"Marcelo Freire","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYDAC5gMMDAkMDHKMDQwMB4jTwpYA1mJMohYgSGwg2l26bbwPHzzcY5Pe3H7G8ABDxT07gnrNjrEbGyQ8S8tt7MkxOMBwpjiZsJb7bWwSCQcO5zbOYEs4wNiWkEzQYWbH2Nh/JBz4n85IihY2hoQDBxIYZzAfAGmxI0YLM9BhyYaNPclAjWcSEojRwvjxxwE7ecP2g80fPlQk2BPUAgeGDQzgOCUhguShNAm2jIJRMApGwUgBAIMgQE6uYEgtAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-4906-7698","institution":"J. 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To address this critical gap, we employed a high-resolution, multi-modal approach—combining RNAscope, GeoMx Digital Spatial Profiling (DSP), spatial transcriptomics, and multiplex immunofluorescence—on 25 terminal ileum and left colon biopsies from a clinical cohort of 8 LC participants and 5 healthy controls. We confirmed the persistence of SARS-CoV-2 Spike transcript and protein in the gut tissue of all LC cases and controls tested. Yet, comparison of Spike-positive (Spike+) regions in LC versus healthy control colon tissues revealed a differential, symptomatic state-associated signature, with 57 differentially expressed genes (DEGs) (26 upregulated, 31 downregulated), revealing genes that disrupt the immune response in LC subjects. LC colon Spike+ regions demonstrated increased expression of AQP8 and other absorptive-related genes (SLC26A3, SLC26A2, and CLCA4) which are involved with Chron’s disease along with transcripts involved in tumorigenesis (GUCA2A, S100P, TSPAN1). Simultaneous downregulation of key homeostatic chemokines (CXCL13, CCL19, CCL21), and other transcripts reported to exhibit low expression in colorectal cancers (TMEM88B, NIBAN3, DMBT1), suggesting a paradox of epithelial tissue stress yet dysfunctional immune trafficking. Further analysis comparing Spike+ versus Spike- regions within LC colon tissue demonstrated an active, localized, antigen-driven immune microenvironment, identifying 122 DEGs (82 upregulated, 40 downregulated), including tumorigenesis genes. Cellular deconvolution of Spike+ regions revealed a statistically significant focal enrichment of myeloid-derived cells (macrophages, non-classical/intermediate monocytes), plasma cells, and regulatory T cells, coupled with significant enrichment in T-cell-related pathways, including ″Antigen processing and presentation,″ and ″Th1/Th2/Th17 cell differentiation″. The ileum displayed a similar, though less pronounced, signature, demonstrating these statistically significant findings are specific to the colon of LC subjects. In contrast, corresponding Spike+ vs. Spike- analysis in healthy control colon tissues showed a more modest transcriptional response with 38 DEGs. Our data provide robust evidence that persistent SARS-CoV-2 Spike protein detection in the gut is not immunologically inert. 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