Dendritic cell targeting virus-like particle delivers mRNA for in vivo immunization

Dendritic cell targeting virus-like particle delivers mRNA for in vivo immunization | 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 Dendritic cell targeting virus-like particle delivers mRNA for in vivo immunization Di Yin, Sikai Ling, Xiaolong Tian, Yang Li, Zhijue Xu, Hewei Jiang, and 15 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1096471/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract mRNA vaccine was approved clinically in 2020. Future development includes delivering mRNA to dendritic cells (DCs) specifically to improve effectiveness and avoid off-target cytotoxicity. Here, we developed virus-like particles (VLPs) as a DC tropic mRNA vaccine vector and showed the prophylactic effects in both SARS-CoV-2 and HSV-1 infection models. The VLP mRNA vaccine elicited strong cytotoxic T cell immunity and durable antibody response with the spike-specific antibodies that lasted for more than 9 months. Importantly, we were able to target mRNA to DCs by pseudotyping VLP with engineered Sindbis virus glycoprotein and found the DC-targeting mRNA vaccine significantly enhanced the titer of antigen-specific IgG, protecting the hACE-2 mice from SARS-CoV-2 infection. Additionally, we showed DC-targeted mRNA vaccine also protected mice from HSV-1 infection when co-delivering the gB and gD mRNA. Thus, the VLP may serve as an in situ DC vaccine and accelerate the further development of mRNA vaccines. Immunology Vaccine Development Biotechnology and Bioengineering SARS-CoV-2 HSV-1 Vaccine mRNA Dendritic cell Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Full Text Additional Declarations Yes there is potential Competing Interest. Y.C. is a consultant and co-founder of BDgene Therapeutics. X.W. is current employee of BDgene Therapeutics. Supplementary Files SupplementaryTablesYinetaltoNBT.docx Supplementary Tables SupplementaryFiguresYinetaltoNBT.docx Supplementary Figures Cite Share Download PDF Status: Posted 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. 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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-1096471","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":66953013,"identity":"00eed535-7b68-4c41-aa07-9c06d6e207c5","order_by":0,"name":"Di Yin","email":"","orcid":"","institution":"Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Di","middleName":"","lastName":"Yin","suffix":""},{"id":66953014,"identity":"315740d7-a082-4887-8326-f9881651af17","order_by":1,"name":"Sikai 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Shanghai","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Yujia","middleName":"","lastName":"Cai","suffix":""}],"badges":[],"createdAt":"2021-11-19 15:26:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1096471/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1096471/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":16080171,"identity":"a7874d83-5ae4-485d-bffd-f8ce88b7bdd9","added_by":"auto","created_at":"2021-12-01 17:50:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":762428,"visible":true,"origin":"","legend":"Design and characterization of VLP-based SARS-CoV-2 mRNA vaccine. a, Construction of mRNA-encoding plasmid which transcribes a MS2 stem loop-containing spike mRNA. The spike mRNA and protein will be packaged into VLP via the RNA-coat protein interaction and self-assembly, respectively. NTD, N-terminal domain; RBD, receptor binding domain; SD1 and SD2, subdomain 1 and 2; FP, fusion peptide; HR1 and HR2, heptad repeat 1 and 2; TM, transmembrane domain; CT, cytoplasmic tail. b, Schematic illustration of the production process of the SARS-CoV-2 vaccine using VLP platform. c, Electron microscopy image of VLP. Scale bar, 100 nm. d, Copy number of spike mRNA in each VLP particle. The copy number was detected by absolute quantification RT-qPCR and normalized to IDLV S-mut (2 copies RNA per virion). e, Western blot analysis of the spike protein in the virion treated with/without PNGase F. IDLV use as a control. 100 ng p24 for each vector. f, Western blot analysis of the spike protein expression. 293T cells were collected 36 h after transfection or transduction. 300 ng p24 virus or VLP used for each well. g. Confocal analysis of spike protein expression. 293T cells were fixed 36 h after transfection or transduction. Images are representative of three independent biological replicates in one experiment. h-j, Innate immune response induced by VLP in THP-1 derived macrophages. Cells were harvested for IFNB1, ISG15 and RIG-I analysis by RT-qPCR 6 h after transduction. 150 ng p24 per well for IDLV S-mut or VLP S-mut. 1.5 μg poly I:C per well as positive controls. ***P\u003c 0.0001 (h-j). S represents spike. S-mut represents mutant spike. Data and error bars represent mean ± s.e.m.; one-way ANOVA with Dunnett’s post hoc tests were performed; n.s.=non-significant.","description":"","filename":"ScreenShot20211201at12.12.54PM.png","url":"https://assets-eu.researchsquare.com/files/rs-1096471/v1/90f220270b2e6a87baa4fadc.png"},{"id":16080125,"identity":"d1c765f8-32a2-4997-b259-a6babe91f7d7","added_by":"auto","created_at":"2021-12-01 17:47:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":673344,"visible":true,"origin":"","legend":"VLP mRNA induces robust and durable spike-specific antibody responses. a, Schematic illustration of the working plan (n=5). The sera were collected 14 days after footpad VLP injection for further analysis. b, ELISA analysis of spike specific IgG. ***P\u003c 0.0001. c-f, Neutralization activity of vaccinated sera evaluated by luciferase assay (c and f), confocal microscopy (d) and plaque assay(e). A firefly luciferase-encoding pseudovirus, GFP-expressing SARS-CoV-2 pseudovirus and live SARS-CoV-2 (USA-WA1/2020) was used, respectively, to transduce Huh-7 or Vero E6 cells. *P= 0.0260 (c). Images are representative of three independent biological replicates in one experiment (d). g and h, Antibody changes in short-term (g) and long-term (h) follow-up vaccination. Mice were immunized with 1.5 μg VLP S-mut via footpad injection, sera were collected at the indicated time for IgG ELISA. Data and error bars represent mean ± s.e.m.; unpaired two-tailed student’s t-tests (b and c); two-tailed Wilcoxon matched-pairs 1 signed-rank test (f).","description":"","filename":"ScreenShot20211201at12.13.13PM.png","url":"https://assets-eu.researchsquare.com/files/rs-1096471/v1/4dd7aa16a210c160849cf7ff.png"},{"id":16080121,"identity":"fb2c2448-7bf7-4f26-9ae4-54ddfb4e34e3","added_by":"auto","created_at":"2021-12-01 17:47:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":769400,"visible":true,"origin":"","legend":"Linear epitope landscape in the VLP mRNA vaccinated mice. a, Representativ images of spike peptide microarray. S1 protein and RBD were included in the microarray as controls. Highly frequent positive peptides were labeled. b. Antibody responses against S1 protein or RBD in vaccinated mice. Signal intensity was averaged fluorescent intensity of tinplated spots for each array. ***P\u003c 0.0001. c, Heatmap of antibody responses against peptides. The gray grid indicates a negative response. d and e, Analysing the epitopes of VLP induced spike-specific antibodies on spike protein. 6 mice were used for each group, 1.5 μg VLP S-mut or 50 μL PBS were injected via footpad into each mouse. Data and error bars represent mean ± s.e.m.; unpaired two-tailed student’s t-tests.","description":"","filename":"ScreenShot20211201at12.13.26PM.png","url":"https://assets-eu.researchsquare.com/files/rs-1096471/v1/b74b52aab9a9954d1f35b8b2.png"},{"id":16080124,"identity":"03225a1e-2e3e-476e-968b-2b172c07644b","added_by":"auto","created_at":"2021-12-01 17:47:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":546374,"visible":true,"origin":"","legend":"DC-targeting VLP-mRNA vaccine induced enhanced spike-specific IgG and T cell immune response. a, Illustration of the production process of the DC-specific VLP-mRNA vaccine. b, Evaluating the DC-specificity of SV-G pseudotyped VLP. 100 ng p24 GFP mRNA-carrying VLP pseudotyped by SV-G and VSV-G, respectively, were transduced to 4x104 DC 2.4 or HeLa cells. Three days later, the transduction efficiency was measured by flow cytometry analysing the GFP expression. ***P =0.0003 of DC2.4 and ***P\u003c 0.0001 of Hela for SV-G VLP versus VSV-G VLP. c, The working plan for analysing VLP mRNA elicited the humoral and cellular immune responses. d and e, ELISA analysis of spike specific and p24 specific IgG. Serum was collected at 14 days post-immunization (1.5 μg p24 VLP per mouse, n=4 mice). *P = 0.0286 for all groups (d). *P = 0.0286 for Mock versus VSV-G and Mock versus SV-G, *P = 0.0571 for VSV-G versus SV-G (e). f-h, Quantification of the number of IFN-γ, TNF-α and IL-6 spot-forming cells isolated from the spleen after stimulation with spike peptide pool. *P = 0.0286 for all groups (f-h). Representative images of ELISPOT wells showed on left. Images are representative of three independent biological replicates in one experiment. Data and error bars represent mean ± s.e.m.; unpaired two-tailed Mann-Whitney tests (d, e, f-h) ; n.s.=non-significant.","description":"","filename":"ScreenShot20211201at12.13.37PM.png","url":"https://assets-eu.researchsquare.com/files/rs-1096471/v1/387377bda1b5589d38c42415.png"},{"id":16080118,"identity":"7eb0ca52-887c-4a62-9b01-c261f0efcef2","added_by":"auto","created_at":"2021-12-01 17:47:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1045801,"visible":true,"origin":"","legend":"DC-specific VLP-mRNA vaccine efficiently protected hACE2 transgenic mice from the SARS-CoV-2 challenge. a, Scheme of vaccination and challenge. 1.5 μg p24 SV-G VLP or 50 μL PBS were immunized by footpad injection (n=6), and boosted at 14 days post prime immunization. Mice were challenged with 105 TCID50 of SARS-CoV-2 at 14 days post boos immunization by intranasal administration. All mice were euthanized at 3 d.p.i. b, Neutralizatio activity of vaccinated sera against live SARS-CoV-2 (USA-WA1/2020). c, The percentage of mic weight change after infection. *P = 0.0253. d and e, Viral loads in lung and trachea detected by RT-qPCR. f, Confocal analysis of SARS-CoV-2 in the lung. **P = 0.0042 (d) and ***P \u003c 0.0001 (c). g, Lung histopathology analysis by hematoxylin and eosin (red arrow, inflammatory cell infiltration; blue arrow, alveolar destruction). Each image is a representative of a group of 4 mice (f and g). Data and error bars represent mean ± s.e.m.; unpaired two-tailed student’s t-tests (c-e).","description":"","filename":"ScreenShot20211201at12.13.53PM.png","url":"https://assets-eu.researchsquare.com/files/rs-1096471/v1/9f84dc8896517f90fa3c1d51.png"},{"id":16080174,"identity":"964fd553-678e-4b52-996c-7ab69245e261","added_by":"auto","created_at":"2021-12-01 17:50:22","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":882962,"visible":true,"origin":"","legend":"DC-specific VLP-mRNA co-delivering gB1 and gD1 efficiently protected mice from the HSV-1 infection. a, Schematic illustration of the production process of the VLP gB1-gD1 vaccine. b, Flowchart for analysing the effectiveness of VLP gB1-gD1 vaccination against HSV-1 infection. Six-week-old C57BL/6 mice (n=4) were immunized with 2 μg p24 VLP mRNA vaccine at day 0 and day 14, respectively. c and d, Neutralization activity against live HSV-1 and HSV-2. n=4 mice. **P = 0.0032 for Prime versus NC, **P = 0.0026 for Boost versus NC and *P = 0.0109 for Prime versus Boost (c). *P = 0.0365 for Prime versus NC, *P = 0.0309 for Boost versus NC (d). e, Representative images of skin at 2 d.p.i. and 5 d.p.i. Each image is representative of four mice in one experiment. f and g, Plaque assay and qPCR analysis of the HSV-1 replication in the skin at 6 d.p.i. *P = 0.0113 (f), *P = 0.0147 (g). h and i, Plaque assay and qPCR analysis of the HSV-1 replication in the DRG at 6 d.p.i. *P = 0.0437 (h), **P = 0.0088 (i). j, HE analysis of skin histopathology at 6 d.p.i. k, IHC analysis of CD4+ and CD8+ T cells infiltration in the skin 6 days after infection. e, epidermis; d, dermis; m, muscle. Each image is a representative of four mice in one experiment (e, j and k). Data and error bars represent mean ± s.e.m.; unpaired two-tailed Student’s t-tests (c, d, f- i); n.s.=non-significant. 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X.W. is current employee of BDgene Therapeutics.","formattedTitle":"Dendritic cell targeting virus-like particle delivers mRNA for in vivo immunization","fulltext":[{"header":"Full Text","content":"This preprint is available for \u003ca href='/article/rs-1096471/latest.pdf' target='_blank'\u003edownload as a PDF\u003c/a\u003e."}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"SARS-CoV-2, HSV-1, Vaccine, mRNA, Dendritic cell","lastPublishedDoi":"10.21203/rs.3.rs-1096471/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1096471/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"mRNA vaccine was approved clinically in 2020. Future development includes delivering mRNA to dendritic cells (DCs) specifically to improve effectiveness and avoid off-target cytotoxicity. Here, we developed virus-like particles (VLPs) as a DC tropic mRNA vaccine vector and showed the prophylactic effects in both SARS-CoV-2 and HSV-1 infection models. The VLP mRNA vaccine elicited strong cytotoxic T cell immunity and durable antibody response with the spike-specific antibodies that lasted for more than 9 months. Importantly, we were able to target mRNA to DCs by pseudotyping VLP with engineered Sindbis virus glycoprotein and found the DC-targeting mRNA vaccine significantly enhanced the titer of antigen-specific IgG, protecting the hACE-2 mice from SARS-CoV-2 infection. Additionally, we showed DC-targeted mRNA vaccine also protected mice from HSV-1 infection when co-delivering the gB and gD mRNA. Thus, the VLP may serve as an in situ DC vaccine and accelerate the further development of mRNA vaccines.","manuscriptTitle":"Dendritic cell targeting virus-like particle delivers mRNA for in vivo immunization","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-12-01 17:47:20","doi":"10.21203/rs.3.rs-1096471/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bc5affa1-cff4-4532-ab6e-815a65aa6254","owner":[],"postedDate":"December 1st, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":8878376,"name":"Immunology"},{"id":8878377,"name":"Vaccine Development"},{"id":8878378,"name":"Biotechnology and Bioengineering"}],"tags":[],"updatedAt":"2021-12-29T10:00:56+00:00","versionOfRecord":[],"versionCreatedAt":"2021-12-01 17:47:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1096471","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1096471","identity":"rs-1096471","version":["v1"]},"buildId":"rHA-KDH7Qsr4HCuvH75dn","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}