Antibodies Targeting the CCHFV Nucleocapsid Protein Require TRIM21 for Protection

Antibodies Targeting the CCHFV Nucleocapsid Protein Require TRIM21 for Protection | 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 Antibodies Targeting the CCHFV Nucleocapsid Protein Require TRIM21 for Protection David Hawman, Shanna Leventhal, Kimberly Meade-White, Carl Shaia, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3915320/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Oct, 2024 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Crimean-Congo Hemorrhagic Fever Virus (CCHFV) is a negative-sense RNA virus spread by Hyalomma genus ticks across Europe, Asia, and Africa. CCHF disease begins as a non-specific febrile illness which may progress into a severe hemorrhagic disease and there are currently no widely approved or highly efficacious interventions available. Recently, we reported a self-replicating, alphavirus-based RNA vaccine which expresses the CCHFV nucleoprotein and is protective against lethal CCHFV disease in mice. This vaccine induces high titers of non-neutralizing anti-NP antibodies and is protective even in mice with constitutive knock-out of Fc-gamma receptors or complement, indicating these mechanisms are not required for protection. Instead, vaccinated mice deficient in the intracellular Fc-receptor TRIM21 despite having robust CCHFV-specific immunity, were unable to control the infection. We also show that passive transfer of NP-immune sera confers significant and TRIM21-dependent protection against lethal CCHFV challenge. Together our data identifies TRIM21 as the Fc effector function of protective antibodies against the CCHFV NP and provides mechanistic insight into how vaccines against CCHFV confer protection. Health sciences/Diseases/Infectious diseases/Viral infection Biological sciences/Microbiology/Virology Biological sciences/Microbiology/Vaccines/RNA vaccines Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Full Text Additional Declarations Yes there is potential Competing Interest. J.E. has equity interest in HDT Bio, and is a co-inventor on U.S. patent application no. 62/993,307 “Compositions and methods for delivery of RNA” pertaining to formulations for RNA delivery. DWH, JE and HF are inventors on U.S. patent application number 63/365,015 “Replicating RNA vaccine for Crimean-Congo hemorrhagic fever virus” regarding the repRNA for use against CCHFV. Supplementary Files SuppFigs.pdf Supplemental Figure 1. repNP vaccination is immunogenic in several antibody effector function deficient mice. WT C57BL6/J or B6NTac mice, FcγR-/-, C3-/- and WT mice depleted of NK cells were vaccinated with 1ug of Sham or repNP RNA on day -28 relative to lethal CCHFV challenge. On D0, (a) FcγR-/- and control, (b) C3-/- and control, and (c) WT mice for NK depletion study were evaluated for CCHFV-specific antibody response via whole virion IgG ELISA and cellular immune response via (d) IFNу ELISpot shown as cumulative responses against peptides spanning the entire CCHFV NP or NP Pool 4. On day 9 p.i., WT mice depleted of NK cells were analyzed for (e) NK cell depletion via flow cytometry. Dashed lines indicate limit of detection. Data shown as mean plus standard deviation. Significance was calculated using one-way ANOVA; **P < 0.01. Supplemental Figure 2. repNP vaccination is immunogenic in TRIM21-/- mice. WT C57BL6/J or TRIM21-/- mice were vaccinated with 1ug of Sham or repNP RNA on day -28 relative to lethal CCHFV challenge. On D0, groups of mice were evaluated for antibody responses via (a) whole virion IgG ELISA and (b) isotype/subtype whole virion IgG ELISA. Cellular immune responses were evaluated via (c) IFNу ELISpot against CCHFV NP peptide pools. Dashed lines indicate limit of detection. Significance was calculated using one-way ANOVA; *P < 0.05. Data shown as mean plus standard deviation. Supplemental Figure 3. repNP vaccination protects WT but not TRIM21-/- mice from spleen pathology. 200X magnification of spleen pathology from (a-b) WT sham vaccinated mice, (c-d) TRIM21-/- sham vaccinated mice, (e-f) WT repNP vaccinated mice, and (g-h) TRIM21-/- repNP vaccinated mice with (left) HE and (right) anti-CCHF IHC reactivity staining. Spleen samples from sham vaccinated and TRIM21-/- repNP vaccinated mice show reduced and apoptotic white pulp with necrotic cellular debris throughout the red pulp. Red pulp mononuclear cells (macrophages) and necrotic debris are immunoreactive. Spleen samples from WT repNP vaccinated mice are normal. Supplemental Figure 4. Gating strategy for FLOW cytometry analyses of CD4+ and CD8+ T-cell Depletions. On D5 p.i., groups of mice (N=6) were euthanized for analyses of T-cell populations in the spleen. (a) Cells were gated on FSC and SSC to exclude debris and doublets. Dead cells were excluded via ZombieAqua viability dye. T-cells were gated by CD3+, CD45+ and CD4+ or CD8+. Cite Share Download PDF Status: Published Journal Publication published 25 Oct, 2024 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-3915320","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":271389191,"identity":"4c38eab2-5b5d-4a3b-980f-d58d44396273","order_by":0,"name":"David Hawman","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA40lEQVRIiWNgGAWjYDCCA0DEAyJB4AODRAJpWhhnEKuFAaaFmYeBgbAWvuNnDA+8Ybgjb3Dt8LPHtjss8hj4Fx+TwKdF8kyOwcE5DM8MN9xOMzfOPSNRzCDxLA2vFoMDaQmHeRgOM267nWAmndsmkdggccbYAK+W88/AWuy33U7/Jm1JlJYbyQdAWhK33c4xk2YEaeHvMXyA1y83Hh84OMfgcPL+2zllkr1ALW0SbIl4tfCdT2z+8KbisO3M2enbJH621SX28x8+cACfFqjzkNhsxMQmGuAnwo5RMApGwSgYUQAA8mhTzh4hOLUAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-8233-8176","institution":"NIAID/NIH","correspondingAuthor":true,"prefix":"","firstName":"David","middleName":"","lastName":"Hawman","suffix":""},{"id":271389192,"identity":"e0d37652-7c30-4b7f-89af-7e6d0abdc56c","order_by":1,"name":"Shanna Leventhal","email":"","orcid":"","institution":"NIAID","correspondingAuthor":false,"prefix":"","firstName":"Shanna","middleName":"","lastName":"Leventhal","suffix":""},{"id":271389193,"identity":"c1b654b2-f4f1-4f5e-9e82-cd097e2b9f5d","order_by":2,"name":"Kimberly Meade-White","email":"","orcid":"","institution":"Rocky Mountain Laboratories, NIAID/NIH","correspondingAuthor":false,"prefix":"","firstName":"Kimberly","middleName":"","lastName":"Meade-White","suffix":""},{"id":271389194,"identity":"e20f6bf4-5dde-4b4c-9127-41531d386433","order_by":3,"name":"Carl Shaia","email":"","orcid":"https://orcid.org/0000-0001-8907-8821","institution":"Rocky Mountain Veterinary Branch, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health","correspondingAuthor":false,"prefix":"","firstName":"Carl","middleName":"","lastName":"Shaia","suffix":""},{"id":271389195,"identity":"bcb3cb76-159b-4532-84b5-0d7d08503ff2","order_by":4,"name":"Justin Murray","email":"","orcid":"","institution":"NIAID/NIH","correspondingAuthor":false,"prefix":"","firstName":"Justin","middleName":"","lastName":"Murray","suffix":""},{"id":271389196,"identity":"0b4c1698-5be6-46b1-b270-8b11f16112cb","order_by":5,"name":"Evan Mihalakakos","email":"","orcid":"","institution":"NIAID/NIH","correspondingAuthor":false,"prefix":"","firstName":"Evan","middleName":"","lastName":"Mihalakakos","suffix":""},{"id":271389197,"identity":"0752d006-3de9-436f-b92f-c9ad6eaf709b","order_by":6,"name":"Troy Hinkley","email":"","orcid":"https://orcid.org/0000-0003-4833-0255","institution":"HDT Bio","correspondingAuthor":false,"prefix":"","firstName":"Troy","middleName":"","lastName":"Hinkley","suffix":""},{"id":271389198,"identity":"f7d7e63e-878d-42c8-a50a-8be03aae39f5","order_by":7,"name":"Jesse Erasmus","email":"","orcid":"","institution":"HDT Bio Corp","correspondingAuthor":false,"prefix":"","firstName":"Jesse","middleName":"","lastName":"Erasmus","suffix":""},{"id":271389199,"identity":"8c46aabc-09f9-4517-8077-3d8da051ee73","order_by":8,"name":"Heinz Feldmann","email":"","orcid":"https://orcid.org/0000-0001-9448-8227","institution":"Laboratory of Virology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health","correspondingAuthor":false,"prefix":"","firstName":"Heinz","middleName":"","lastName":"Feldmann","suffix":""}],"badges":[],"createdAt":"2024-01-31 22:50:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3915320/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3915320/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-024-53362-7","type":"published","date":"2024-10-25T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":51155662,"identity":"01585a93-4564-41aa-9d3e-583af2f948de","added_by":"auto","created_at":"2024-02-15 05:48:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":89645,"visible":true,"origin":"","legend":"\u003cp\u003eAdoptive transfer of repNP vaccinated mouse sera increases survival in naïve CCHFV infected mice. Sera stocks for adoptive transfer were confirmed to have CCHFV-specific antibodies via (a) whole virion IgG ELISA and isotype/subtype whole virion IgG ELISA. Naïve WT C57BL6/J mice were treated with sera from repNP or sham vaccinated mice on day -1 (1tx) or days 0 and +3 (2tx) relative to lethal challenge with 100 TCID50 CCHFV strain UG3010. Mice (N=8) were (b) weighed daily and monitored for (c) survival until day 14 p.i.. Dashed lines indicate limit of detection. Significance was calculated using one-way ANOVA; ns P \u0026gt; 0.05. Data shown as mean plus standard deviation.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-3915320/v1/82dbf1c3a145da7f6756ccbf.png"},{"id":51155661,"identity":"9b0b1594-3376-4409-9889-2382c4bb5799","added_by":"auto","created_at":"2024-02-15 05:48:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":199820,"visible":true,"origin":"","legend":"\u003cp\u003erepNP vaccination is efficacious in the absence of Fcγ Receptors, Complement, and NK cells. WT C57BL6/J or B6NTac mice, FcγR-/-, C3-/- and WT mice depleted of NK cells were (a-b) vaccinated with 1ug of Sham or repNP RNA on day -28 relative to lethal CCHFV challenge. (b) WT mice were depleted of NK cells on Day -2, 1, 4, 7, and 10 relative to CCHFV challenge by IP treatment with NK1.1 antibody. On D0, groups of mice were treated with MAR1-5A3 antibody and infected with a lethal dose of 100 TCID50 CCHFV strain UG3010. Mice (N=8) were (c) weighed daily and monitored for (d) survival until day 14 post-infection (p.i.). On D5 p.i., groups of mice (N=6) were euthanized and evaluated for (e) viral genome copies via qRT-PCR and (f) infectious virus via TCID50 in the blood, liver, and spleen. WT repNP mice are pooled C57BL/6 and B6NTac mice vaccinated with repNP RNA. Sham mice are pooled C57BL/6, B6NTac, FcγR-/-, and C3-/- mice vaccinated with Sham RNA. Dashed lines indicate limit of detection. Significance was calculated using one-way ANOVA; ns P \u0026gt; 0.05, *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001. Data shown as mean plus standard deviation.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-3915320/v1/a25b67edf0caa5f34c206210.png"},{"id":51156296,"identity":"cc5b7423-98a4-461b-bb9c-c13fcb7d8ee4","added_by":"auto","created_at":"2024-02-15 05:56:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":175729,"visible":true,"origin":"","legend":"\u003cp\u003erepNP vaccination fails to protect TRIM21-/- mice. WT C57BL6/J or TRIM21-/- mice were (a) vaccinated with 1ug of Sham or repNP RNA on day -28 relative to lethal CCHFV challenge. On D0, groups of mice were treated with MAR1-5A3 antibody and infected with a lethal dose of 100 TCID50 CCHFV strain UG3010. Mice (N=8) were (b) weighed daily and monitored for (c) survival until day 14 p.i.. On D5 p.i., groups of mice (N=6) were euthanized and evaluated for (d) viral genome copies via qRT-PCR, and (e) infectious virus via TCID50 in the blood, liver, and spleen. Dashed lines indicate limit of detection. Significance was calculated using one-way ANOVA; **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001. Data shown as mean plus standard deviation.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-3915320/v1/71da971a14854e18dba6dbfe.png"},{"id":51156298,"identity":"71a663c8-93e5-4c6c-bbfe-1437438da534","added_by":"auto","created_at":"2024-02-15 05:56:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1324297,"visible":true,"origin":"","legend":"\u003cp\u003erepNP vaccination protects WT but not TRIM21-/- mice from liver pathology. 200X magnification of liver pathology from (a-b) WT sham vaccinated mice, (c-d) TRIM21-/- sham vaccinated mice, (e-f) WT repNP vaccinated mice, and (g-h) TRIM21-/- repNP vaccinated mice with (left) HE and (right) anti-CCHF IHC reactivity staining. Liver samples from sham vaccinated and TRIM21-/- repNP vaccinated mice show clusters of necrotic cellular debris multifocally dispersed throughout the hepatic plates. Sinusoidal mononuclear cells (Kupffer cells) and necrotic hepatocytes are immunoreactive. The liver samples from WT repNP vaccinated mice are normal.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-3915320/v1/a4a7ae1ce05c44ba9ad42e79.png"},{"id":51156297,"identity":"6a5c5b1a-efd2-49be-b2b4-a8e9bc1b571b","added_by":"auto","created_at":"2024-02-15 05:56:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":196201,"visible":true,"origin":"","legend":"\u003cp\u003eWT mice depleted of CD4+ and CD8+ T-cells are protected by repNP vaccination. On D5 p.i., groups of mice (N=6) from Figure 3 were euthanized and evaluated for cellular immune responses to infection via (a) IFNу ELISpot shown as cumulative responses against peptides spanning the entire CCHFV NP. For T-cell depletion study, WT C57BL6/J mice were (b) vaccinated with Sham or repNP RNA on day -28 relative to lethal CCHFV challenge. On days -5, -2, and +5 relative to CCHFV challenge, mice were treated with isotype or αCD4 and αCD8 antibody to deplete mice of T-cell populations. On D0, groups of mice were treated with MAR1-5A3 antibody and infected with a lethal dose of 100 TCID50 CCHFV strain UG3010. Mice (N=8) were (c) weighed daily and monitored for (d) survival until day 14 p.i.. On D5 p.i., groups of mice (N=6) were evaluated for (e) depletion of CD4+ and CD8+ T-cell populations via Flow Cytometry, (f) viral genome copies via qRT-PCR and (g) infectious virus via TCID50 in the blood, liver, and spleen. Dashed lines indicate limit of detection. Significance was calculated using one-way ANOVA; ns P \u0026gt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001. Data shown as mean plus standard deviation. Gating strategy for FLOW cytometry analyses is shown in Supplemental Figure 4a.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-3915320/v1/4a1915d264b0e4eca2ba7437.png"},{"id":51155664,"identity":"9aefbe97-2a03-4b60-ba6f-6afaf0bc812f","added_by":"auto","created_at":"2024-02-15 05:48:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":190208,"visible":true,"origin":"","legend":"\u003cp\u003eNP-immune sera protects WT but not TRIM21-/- mice from lethal CCHFV infection and blocks CCHFV infection in vitro. Sera stocks for adoptive transfer were the same as described in Figure 1. Naïve WT C57BL6/J and TRIM21-/- mice were treated with 400ul of sham or NP-immune sera (a) 6 hours prior to lethal challenge with 100 TCID50 CCHFV strain UG3010. Mice (N=8) were (b) weighed daily and monitored for (c) survival until day 14 p.i.. Data shown as mean plus standard deviation. To investigate antibody-dependent intracellular neutralization, L929 cells were electroporated with antibody and efficiency of electroporation was verified via (d) FLOW cytometry measuring internalization of control anti-mouse AF488 antibody with and without electroporation (EP); cells were gated by AF488- or AF488+. Next, L929 cells were electroporated with (e) mouse sham or NP-immune sera, as used in adoptive transfer studies, and infected with MA-CCHFV or CCHFV strain UG3010. Viral growth was monitored via qRT-PCR for 4 days post-infection. Study was performed twice, each with three technical replicates. (f) Pre-immune and NP-immune sera from cynomolgus macaques vaccinated with our repNP vaccine was pooled and evaluated for CCHFV-specific antibody via (f) ELISA and then electroporated into L929 cells to assess ability to neutralize (g) MA-CCHFV or CCHFV UG3010 infection as measured via qRT-PCR. Study performed once with three technical replicates. Significance was calculated using one-way ANOVA; ns P \u0026gt; 0.05, ****P \u0026lt; 0.0001. Data shown as mean plus standard deviation. Gating strategy for FLOW cytometry analyses is shown in Supplemental Figure 4b.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-3915320/v1/19c1a96d83c3651116ffc12d.png"},{"id":67530104,"identity":"97bb4fe2-bb10-4f7e-88cb-21281dab2dcc","added_by":"auto","created_at":"2024-10-26 07:07:06","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1072520,"visible":true,"origin":"","legend":"","description":"","filename":"TRIM21Submission.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3915320/v1_covered_032be5a4-31e4-4c21-ac2e-2f8e7c302e4d.pdf"},{"id":51155666,"identity":"1022875d-0faf-4bc5-af34-79fafd7ac4c3","added_by":"auto","created_at":"2024-02-15 05:48:07","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13032095,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Figure 1.\u003c/strong\u003e repNP vaccination is immunogenic in several antibody effector function deficient mice. WT C57BL6/J or B6NTac mice, FcγR-/-, C3-/- and WT mice depleted of NK cells were vaccinated with 1ug of Sham or repNP RNA on day -28 relative to lethal CCHFV challenge. On D0, (a) FcγR-/- and control, (b) C3-/- and control, and (c) WT mice for NK depletion study were evaluated for CCHFV-specific antibody response via whole virion IgG ELISA and cellular immune response via (d) IFNу ELISpot shown as cumulative responses against peptides spanning the entire CCHFV NP or NP Pool 4. On day 9 p.i., WT mice depleted of NK cells were analyzed for (e) NK cell depletion via flow cytometry. Dashed lines indicate limit of detection. Data shown as mean plus standard deviation. Significance was calculated using one-way ANOVA; **P \u0026lt; 0.01.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Figure 2.\u003c/strong\u003e repNP vaccination is immunogenic in TRIM21-/- mice. WT C57BL6/J or TRIM21-/- mice were vaccinated with 1ug of Sham or repNP RNA on day -28 relative to lethal CCHFV challenge. On D0, groups of mice were evaluated for antibody responses via (a) whole virion IgG ELISA and (b) isotype/subtype whole virion IgG ELISA. Cellular immune responses were evaluated via (c) IFNу ELISpot against CCHFV NP peptide pools. Dashed lines indicate limit of detection. Significance was calculated using one-way ANOVA; *P \u0026lt; 0.05. Data shown as mean plus standard deviation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Figure 3.\u003c/strong\u003e repNP vaccination protects WT but not TRIM21-/- mice from spleen pathology. 200X magnification of spleen pathology from (a-b) WT sham vaccinated mice, (c-d) TRIM21-/- sham vaccinated mice, (e-f) WT repNP vaccinated mice, and (g-h) TRIM21-/- repNP vaccinated mice with (left) HE and (right) anti-CCHF IHC reactivity staining. Spleen samples from sham vaccinated and TRIM21-/-\u003c/p\u003e\n\u003cp\u003erepNP vaccinated mice show reduced and apoptotic white pulp with necrotic cellular debris throughout the red pulp. Red pulp mononuclear cells (macrophages) and necrotic debris are immunoreactive. Spleen samples from WT repNP vaccinated mice are normal.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Figure 4.\u003c/strong\u003e Gating strategy for FLOW cytometry analyses of CD4+ and CD8+ T-cell Depletions. On D5 p.i., groups of mice (N=6) were euthanized for analyses of T-cell populations in the spleen. (a) Cells were gated on FSC and SSC to exclude debris and doublets. Dead cells were excluded via ZombieAqua viability dye. T-cells were gated by CD3+, CD45+ and CD4+ or CD8+.\u003c/p\u003e","description":"","filename":"SuppFigs.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3915320/v1/6649c5f9e9fd625bc68f6ceb.pdf"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nJ.E. has equity interest in HDT Bio, and is a co-inventor on U.S. patent application no. 62/993,307 “Compositions and methods for delivery of RNA” pertaining to formulations for RNA delivery. DWH, JE and HF are inventors on U.S. patent application number 63/365,015 “Replicating RNA vaccine for Crimean-Congo hemorrhagic fever virus” regarding the repRNA for use against CCHFV.","formattedTitle":"Antibodies Targeting the CCHFV Nucleocapsid Protein Require TRIM21 for Protection","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":"","lastPublishedDoi":"10.21203/rs.3.rs-3915320/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3915320/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCrimean-Congo Hemorrhagic Fever Virus (CCHFV) is a negative-sense RNA virus spread by Hyalomma genus ticks across Europe, Asia, and Africa. CCHF disease begins as a non-specific febrile illness which may progress into a severe hemorrhagic disease and there are currently no widely approved or highly efficacious interventions available. Recently, we reported a self-replicating, alphavirus-based RNA vaccine which expresses the CCHFV nucleoprotein and is protective against lethal CCHFV disease in mice. This vaccine induces high titers of non-neutralizing anti-NP antibodies and is protective even in mice with constitutive knock-out of Fc-gamma receptors or complement, indicating these mechanisms are not required for protection. Instead, vaccinated mice deficient in the intracellular Fc-receptor TRIM21 despite having robust CCHFV-specific immunity, were unable to control the infection. We also show that passive transfer of NP-immune sera confers significant and TRIM21-dependent protection against lethal CCHFV challenge. Together our data identifies TRIM21 as the Fc effector function of protective antibodies against the CCHFV NP and provides mechanistic insight into how vaccines against CCHFV confer protection.\u003c/p\u003e","manuscriptTitle":"Antibodies Targeting the CCHFV Nucleocapsid Protein Require TRIM21 for Protection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-15 05:48:02","doi":"10.21203/rs.3.rs-3915320/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":"05ea5fb1-04ee-45cb-89de-d7585c749bdf","owner":[],"postedDate":"February 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":28610552,"name":"Health sciences/Diseases/Infectious diseases/Viral infection"},{"id":28610553,"name":"Biological sciences/Microbiology/Virology"},{"id":28610554,"name":"Biological sciences/Microbiology/Vaccines/RNA vaccines"}],"tags":[],"updatedAt":"2024-10-26T07:06:58+00:00","versionOfRecord":{"articleIdentity":"rs-3915320","link":"https://doi.org/10.1038/s41467-024-53362-7","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2024-10-25 04:00:00","publishedOnDateReadable":"October 25th, 2024"},"versionCreatedAt":"2024-02-15 05:48:02","video":"","vorDoi":"10.1038/s41467-024-53362-7","vorDoiUrl":"https://doi.org/10.1038/s41467-024-53362-7","workflowStages":[]},"version":"v1","identity":"rs-3915320","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3915320","identity":"rs-3915320","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}