{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,1,16]],"date-time":"2026-01-16T01:49:01Z","timestamp":1768528141553,"version":"3.49.0"},"posted":{"date-parts":[[2025,12,1]]},"group-title":"Immunology","reference-count":72,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,12,1]],"date-time":"2025-12-01T00:00:00Z","timestamp":1764547200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,12,1]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>The COVID-19 pandemic has demonstrated the detrimental potential of zoonotic coronavirus transmission to human populations. Effective vaccines capable of eliciting immunity to SARS-CoV-2 have been pivotal in mitigating the spread of the virus. In this study, we describe the generation of a non-replicating pseudotyped influenza A virus (S-FLU), where the native haemagglutinin (HA) sequence is replaced with the coding sequence of either a membrane-anchored form (TM) or secretory form (Sec) of the receptor-binding domain (RBD) of the ancestral SARS-CoV-2 Wuhan (S-RBD Wuhan). We showed that both S-RBD-TM and S-RBD-Sec viruses can be generated via reverse genetics and grown to high titre. Intranasal immunisation in mice with S-RBD-TM elicits robust serum binding and neutralisation activity against SARS-CoV-2, superior to S-RBD-Sec. Furthermore, we demonstrate that a heterologous prime-boost immunisation regime in mice with S-RBD-TM Wuhan and S-RBD-TM BM48-31 (a distant Clade 3 SARS-like betacoronavirus (sarbecovirus)) increases antibody breadth against mismatched sarbecoviruses compared to homologous prime-boost with S-RBD-TM Wuhan. These results suggest that S-RBD-TM is a promising intranasal vaccine candidate against SARS-CoV-2 and may offer potential as a broadly protective sarbecovirus vaccine.<\/jats:p>","DOI":"10.1101\/2025.11.28.691177","type":"posted-content","created":{"date-parts":[[2025,12,2]],"date-time":"2025-12-02T03:05:18Z","timestamp":1764644718000},"source":"Crossref","is-referenced-by-count":0,"title":["Development of a Recombinant Single-Cycle Influenza Viral Vector as an Intranasal Vaccine against SARS-CoV-2 and SARS-like Betacoronaviruses"],"prefix":"10.64898","author":[{"given":"Jonathan","family":"Munro","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Diana","family":"Melnyk","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Madeeha","family":"Afzal","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lisa","family":"Schimanski","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alexander A.","family":"Cohen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jennifer R.","family":"Keeffe","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Pamela J.","family":"Bjorkman","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"William S.","family":"James","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3702-0107","authenticated-orcid":false,"given":"Alain R.","family":"Townsend","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8746-8308","authenticated-orcid":false,"given":"Tiong Kit","family":"Tan","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"issue":"7803","key":"2025120316401112000_2025.11.28.691177v1.1","doi-asserted-by":"crossref","first-page":"E7","DOI":"10.1038\/s41586-020-2202-3","article-title":"Author Correction: A new coronavirus associated with human respiratory disease in China","volume":"580","year":"2020","journal-title":"Nature"},{"key":"2025120316401112000_2025.11.28.691177v1.2","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-020-2951-z"},{"key":"2025120316401112000_2025.11.28.691177v1.3","doi-asserted-by":"publisher","DOI":"10.1016\/S0140-6736(22)01585-9"},{"key":"2025120316401112000_2025.11.28.691177v1.4","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-022-04399-5"},{"issue":"13","key":"2025120316401112000_2025.11.28.691177v1.5","doi-asserted-by":"crossref","first-page":"3426","DOI":"10.1016\/j.cell.2021.04.025","article-title":"Transmission, infectivity, and neutralization of a spike L452R SARS-CoV-2 variant","volume":"184","year":"2021","journal-title":"Cell"},{"key":"2025120316401112000_2025.11.28.691177v1.6","doi-asserted-by":"publisher","DOI":"10.1016\/J.CELL.2021.06.020\/ATTACHMENT\/10183911-F97E-4DA5-AE05-227854F207F4\/MMC1.PDF"},{"issue":"7854","key":"2025120316401112000_2025.11.28.691177v1.7","doi-asserted-by":"crossref","first-page":"438","DOI":"10.1038\/s41586-021-03402-9","article-title":"Detection of a SARS-CoV-2 variant of concern in South Africa","volume":"592","year":"2021","journal-title":"Nature"},{"key":"2025120316401112000_2025.11.28.691177v1.8","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-021-03470-x"},{"issue":"7922","key":"2025120316401112000_2025.11.28.691177v1.9","doi-asserted-by":"crossref","first-page":"E24","DOI":"10.1038\/s41586-022-05103-3","article-title":"Author Correction: Sensitivity of SARS-CoV-2 B.1.1.7 to mRNA vaccine-elicited antibodies","volume":"608","year":"2022","journal-title":"Nature"},{"key":"2025120316401112000_2025.11.28.691177v1.10","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMc2207519"},{"key":"2025120316401112000_2025.11.28.691177v1.11","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMc2031364"},{"key":"2025120316401112000_2025.11.28.691177v1.12","doi-asserted-by":"publisher","DOI":"10.1038\/S41591-021-01285-X"},{"key":"2025120316401112000_2025.11.28.691177v1.13","doi-asserted-by":"publisher","DOI":"10.1038\/s41579-018-0118-9"},{"key":"2025120316401112000_2025.11.28.691177v1.14","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-micro-020518-115759"},{"key":"2025120316401112000_2025.11.28.691177v1.15","doi-asserted-by":"publisher","DOI":"10.1126\/science.1118391"},{"key":"2025120316401112000_2025.11.28.691177v1.16","doi-asserted-by":"publisher","DOI":"10.1126\/science.aac8608"},{"key":"2025120316401112000_2025.11.28.691177v1.17","doi-asserted-by":"publisher","DOI":"10.1016\/S0140-6736(03)14630-2"},{"key":"2025120316401112000_2025.11.28.691177v1.18","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa030747"},{"key":"2025120316401112000_2025.11.28.691177v1.19","doi-asserted-by":"publisher","DOI":"10.1016\/S0140-6736(03)13413-7"},{"key":"2025120316401112000_2025.11.28.691177v1.20","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa1211721"},{"key":"2025120316401112000_2025.11.28.691177v1.21","doi-asserted-by":"crossref","first-page":"102026","DOI":"10.1016\/j.tmaid.2021.102026","article-title":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV) coinfection: A unique case series","volume":"41","year":"2021","journal-title":"Travel Med Infect Dis"},{"key":"2025120316401112000_2025.11.28.691177v1.22","doi-asserted-by":"crossref","unstructured":"van Boheemen, S. , et al., Genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans. mBio, 2012. 3(6).","DOI":"10.1128\/mBio.00473-12"},{"key":"2025120316401112000_2025.11.28.691177v1.23","doi-asserted-by":"publisher","DOI":"10.1038\/nature12711"},{"key":"2025120316401112000_2025.11.28.691177v1.24","doi-asserted-by":"crossref","unstructured":"Anthony, S.J. , et al., Further Evidence for Bats as the Evolutionary Source of Middle East Respiratory Syndrome Coronavirus. Mbio, 2017. 8(2).","DOI":"10.1128\/mBio.00373-17"},{"issue":"4","key":"2025120316401112000_2025.11.28.691177v1.25","doi-asserted-by":"crossref","first-page":"562","DOI":"10.1038\/s41564-020-0688-y","article-title":"Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses","volume":"5","year":"2020","journal-title":"Nature Microbiology"},{"key":"2025120316401112000_2025.11.28.691177v1.26","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1517719113"},{"key":"2025120316401112000_2025.11.28.691177v1.27","doi-asserted-by":"publisher","DOI":"10.1007\/s12250-018-0012-7"},{"key":"2025120316401112000_2025.11.28.691177v1.28","doi-asserted-by":"publisher","DOI":"10.1016\/j.vaccine.2023.02.032"},{"key":"2025120316401112000_2025.11.28.691177v1.29","doi-asserted-by":"publisher","DOI":"10.1038\/s41541-022-00597-4"},{"key":"2025120316401112000_2025.11.28.691177v1.30","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2020.11.032"},{"key":"2025120316401112000_2025.11.28.691177v1.31","doi-asserted-by":"crossref","first-page":"93","DOI":"10.1016\/bs.aivir.2019.08.002","article-title":"Structural insights into coronavirus entry","volume":"105","year":"2019","journal-title":"Complementary Strategies to Understand Virus Structure and Function"},{"key":"2025120316401112000_2025.11.28.691177v1.32","doi-asserted-by":"publisher","DOI":"10.1126\/science.abc5902"},{"key":"2025120316401112000_2025.11.28.691177v1.33","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1010148"},{"key":"2025120316401112000_2025.11.28.691177v1.34","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-020-2571-7"},{"key":"2025120316401112000_2025.11.28.691177v1.35","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-020-2456-9"},{"key":"2025120316401112000_2025.11.28.691177v1.36","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-020-2852-1"},{"key":"2025120316401112000_2025.11.28.691177v1.37","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-023-35949-8"},{"key":"2025120316401112000_2025.11.28.691177v1.38","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-020-2349-y"},{"key":"2025120316401112000_2025.11.28.691177v1.39","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-021-03817-4"},{"key":"2025120316401112000_2025.11.28.691177v1.40","doi-asserted-by":"publisher","DOI":"10.1126\/science.abc7424"},{"key":"2025120316401112000_2025.11.28.691177v1.41","doi-asserted-by":"publisher","DOI":"10.1016\/j.celrep.2021.110188"},{"key":"2025120316401112000_2025.11.28.691177v1.42","doi-asserted-by":"publisher","DOI":"10.1128\/jvi.01596-22"},{"issue":"7","key":"2025120316401112000_2025.11.28.691177v1.43","doi-asserted-by":"crossref","first-page":"110208","DOI":"10.1016\/j.isci.2024.110208","article-title":"A cryptic site in class 5 epitope of SARS-CoV-2 RBD maintains highly conservation across natural isolates","volume":"27","year":"2024","journal-title":"iScience"},{"key":"2025120316401112000_2025.11.28.691177v1.44","doi-asserted-by":"publisher","DOI":"10.1099\/jgv.0.001228"},{"key":"2025120316401112000_2025.11.28.691177v1.45","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.01820-12"},{"key":"2025120316401112000_2025.11.28.691177v1.46","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-023-37697-1"},{"key":"2025120316401112000_2025.11.28.691177v1.47","doi-asserted-by":"crossref","unstructured":"Loes, A.N. , et al., Attenuated Influenza Virions Expressing the SARS-CoV-2 Receptor-Binding Domain Induce Neutralizing Antibodies in Mice. Viruses, 2020. 12(9).","DOI":"10.3390\/v12090987"},{"key":"2025120316401112000_2025.11.28.691177v1.48","doi-asserted-by":"crossref","unstructured":"Moser, M.J. , et al., Intranasal Single-Replication Influenza Vector Induces Cross-Reactive Serum and Mucosal Antibodies against SARS-CoV-2 Variants. Vaccines (Basel), 2023. 11(6).","DOI":"10.3390\/vaccines11061063"},{"key":"2025120316401112000_2025.11.28.691177v1.49","doi-asserted-by":"crossref","first-page":"109491","DOI":"10.1016\/j.vetmic.2022.109491","article-title":"An influenza virus vector candidate vaccine stably expressing SARS-CoV-2 receptor-binding domain produces high and long-lasting neutralizing antibodies in mice","volume":"271","year":"2022","journal-title":"Vet Microbiol"},{"key":"2025120316401112000_2025.11.28.691177v1.50","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-020-15562-9"},{"key":"2025120316401112000_2025.11.28.691177v1.51","doi-asserted-by":"crossref","unstructured":"Xiao, J.H. , et al., Characterization of Influenza Virus Pseudotyped with Ebolavirus Glycoprotein. J Virol, 2018. 92(4).","DOI":"10.1128\/JVI.00941-17"},{"key":"2025120316401112000_2025.11.28.691177v1.52","doi-asserted-by":"crossref","first-page":"1608074","DOI":"10.3389\/fimmu.2025.1608074","article-title":"Single-cycle, pseudotyped reporter influenza virus to facilitate evaluation of treatment strategies for avian influenza, Ebola and other highly infectious diseases in vivo","volume":"16","year":"2025","journal-title":"Front Immunol"},{"key":"2025120316401112000_2025.11.28.691177v1.53","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-020-20654-7"},{"key":"2025120316401112000_2025.11.28.691177v1.54","doi-asserted-by":"crossref","first-page":"370","DOI":"10.1016\/j.omtn.2023.01.008","article-title":"Broadly neutralizing aptamers to SARS-CoV-2: A diverse panel of modified DNA antiviral agents","volume":"31","year":"2023","journal-title":"Mol Ther Nucleic Acids"},{"issue":"6","key":"2025120316401112000_2025.11.28.691177v1.55","doi-asserted-by":"crossref","first-page":"112451","DOI":"10.1016\/j.isci.2025.112451","article-title":"Determination of resilience of a panel of broadly neutralizing mAbs to emerging variants of SARS-CoV-2 generated using reverse genetics","volume":"28","year":"2025","journal-title":"iScience"},{"key":"2025120316401112000_2025.11.28.691177v1.56","doi-asserted-by":"crossref","unstructured":"Van Reeth, K. , et al., Heterologous prime-boost vaccination with H3N2 influenza viruses of swine favors cross-clade antibody responses and protection. NPJ Vaccines, 2017. 2.","DOI":"10.1038\/s41541-017-0012-x"},{"key":"2025120316401112000_2025.11.28.691177v1.57","doi-asserted-by":"crossref","unstructured":"Bhatnagar, N. , et al., Heterologous Prime-Boost Vaccination with Inactivated Influenza Viruses Induces More Effective Cross-Protection than Homologous Repeat Vaccination. Vaccines (Basel), 2023. 11(7).","DOI":"10.3390\/vaccines11071209"},{"key":"2025120316401112000_2025.11.28.691177v1.58","doi-asserted-by":"crossref","first-page":"1142394","DOI":"10.3389\/fimmu.2023.1142394","article-title":"Heterologous prime-boost immunisation with mRNA- and AdC68-based 2019-nCoV variant vaccines induces broad-spectrum immune responses in mice","volume":"14","year":"2023","journal-title":"Front Immunol"},{"key":"2025120316401112000_2025.11.28.691177v1.59","doi-asserted-by":"publisher","DOI":"10.1126\/science.abq0839"},{"key":"2025120316401112000_2025.11.28.691177v1.60","doi-asserted-by":"publisher","DOI":"10.1128\/mBio.01487-15"},{"key":"2025120316401112000_2025.11.28.691177v1.61","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.2001086"},{"key":"2025120316401112000_2025.11.28.691177v1.62","doi-asserted-by":"crossref","unstructured":"Kandasamy, M. , et al., Recombinant single-cycle influenza virus with exchangeable pseudotypes allows repeated immunization to augment anti-tumour immunity with immune checkpoint inhibitors. Elife, 2023. 12.","DOI":"10.7554\/eLife.76414"},{"key":"2025120316401112000_2025.11.28.691177v1.63","doi-asserted-by":"publisher","DOI":"10.1126\/science.abc6284"},{"key":"2025120316401112000_2025.11.28.691177v1.64","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1011670"},{"key":"2025120316401112000_2025.11.28.691177v1.65","doi-asserted-by":"publisher","DOI":"10.1038\/s41577-022-00813-1"},{"key":"2025120316401112000_2025.11.28.691177v1.66","doi-asserted-by":"publisher","DOI":"10.1016\/j.coi.2022.102278"},{"key":"2025120316401112000_2025.11.28.691177v1.67","doi-asserted-by":"crossref","unstructured":"Odle, A. , et al., Tissue-resident memory T cells contribute to protection against heterologous SARS-CoV-2 challenge. JCI Insight, 2024. 9(23).","DOI":"10.1172\/jci.insight.184074"},{"key":"2025120316401112000_2025.11.28.691177v1.68","doi-asserted-by":"publisher","DOI":"10.1038\/s41594-020-0480-y"},{"key":"2025120316401112000_2025.11.28.691177v1.69","doi-asserted-by":"publisher","DOI":"10.1126\/science.abd0826"},{"key":"2025120316401112000_2025.11.28.691177v1.70","doi-asserted-by":"crossref","unstructured":"Crawford, K.H.D. , et al., Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays. Viruses, 2020. 12(5).","DOI":"10.3390\/v12050513"},{"key":"2025120316401112000_2025.11.28.691177v1.71","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1309215110"},{"key":"2025120316401112000_2025.11.28.691177v1.72","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2024.07.052"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2025.11.28.691177","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T10:07:20Z","timestamp":1768471640000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2025.11.28.691177"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,12,1]]},"references-count":72,"URL":"https:\/\/doi.org\/10.1101\/2025.11.28.691177","relation":{},"subject":[],"published":{"date-parts":[[2025,12,1]]},"subtype":"preprint"}}