{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,8,9]],"date-time":"2026-08-09T08:41:31Z","timestamp":1786264891477,"version":"build-2736575974"},"posted":{"date-parts":[[2026,8,6]]},"group-title":"Microbiology","reference-count":89,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,8,6]],"date-time":"2026-08-06T00:00:00Z","timestamp":1785974400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,8,6]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>Development of an effective HCV vaccine requires the induction of both broadly neutralizing antibodies (bnAbs) and a robust cellular response. One issue that has arisen is that HCV subunit vaccines have limited immunogenicity, thus requiring multivalent formats in order to elicit a robust anti-HCV immune response. Toward that end, nanoparticle vaccines possess the ability to facilitate a controlled multivalent presentation and trafficking to lymph nodes, where they can interact with both arms of the immune system. Here, we used a soluble, secreted form of E1E2 (sE1E2) to assemble native E1E2 into a nanoparticle platform using a post-purification coupling assembly system. Nanoparticles were assembled by purifying sE1E2 containing a C-terminal SpyTag and an mi3-SpyCatcher fusion separately and covalently coupling the components via incubation. Free sE1E2-SpyTag was removed from nanoparticle preparations via gel filtration. The sE1E2-mi3 nanoparticles are fully competent to bind conformation-dependent bnAbs, indicating retention of a native assembly in the nanoparticle format. Electron microscopy analysis showed a clear incorporation of sE1E2 on the surface of the nanoparticle. Immunogenicity of sE1E2-mi3 nanoparticles was examined relative to sE1E2 alone and membrane-bound E1E2 (mbE1E2) following inoculation of groups of CD1 mice. Assessment of the immunogenicity of the sE1E2-mi3 nanoparticles showed that the nanoparticle assembly has a similar immunogenicity profile to that of mbE1E2 after only a prime and one boost, and overall superior to sE1E2. This proof-of-principle study sets the stage for further exploration of nanoparticles and other multivalent platforms for the development of E1E2-based vaccines.<\/jats:p>\n                <jats:sec>\n                  <jats:title>Importance<\/jats:title>\n                  <jats:p>Hepatitis C virus infects approximately 50 million people, and at present no effective HCV vaccine exists. Due to the high sequence variability of HCV and the resulting difficulty in developing a vaccine that elicits a broadly neutralizing response, multiple efforts are underway to enhance the immunogenicity of HCV vaccine candidates. In this study, we incorporated native soluble, secreted E1E2 (sE1E2) into a 60-mer nanoparticle via the SpyTag-SpyCatcher system and covalent isopeptide bond attachment using the purified components. These nanoparticles are antigenically intact and elicit a neutralizing antibody response at an earlier time point in the immunization regimen than the corresponding subunit vaccine. These studies show that a well-characterized sE1E2 platform compatible with multiple genotypes can be coupled to nanoparticles for use as a vaccine candidate.<\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.08.05.743069","type":"posted-content","created":{"date-parts":[[2026,8,7]],"date-time":"2026-08-07T04:50:58Z","timestamp":1786078258000},"source":"Crossref","is-referenced-by-count":0,"title":["Incorporation of native HCV E1E2 into a nanoparticle vaccine platform"],"prefix":"10.64898","author":[{"given":"Liudmila","family":"Kulakova","sequence":"first","affiliation":[{"name":"University of Maryland Institute for Bioscience and Biotechnology Research, Rockville, MD 20850, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sarah","family":"Jeong","sequence":"additional","affiliation":[{"name":"University of Maryland Institute for Bioscience and Biotechnology Research, Rockville, MD 20850, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"DongXiu","family":"Zhang","sequence":"additional","affiliation":[{"name":"University of Maryland Institute for Bioscience and Biotechnology Research, Rockville, MD 20850, USA"},{"name":"Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaoran","family":"Shang","sequence":"additional","affiliation":[{"name":"University of Maryland Institute for Bioscience and Biotechnology Research, Rockville, MD 20850, USA"},{"name":"Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD, USA"},{"name":"The Center for Biomolecular Therapeutics (CBT), University of Maryland School of Medicine, Baltimore, MD, USA 21201"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kinlin L.","family":"Chao","sequence":"additional","affiliation":[{"name":"University of Maryland Institute for Bioscience and Biotechnology Research, Rockville, MD 20850, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexander","family":"Marin","sequence":"additional","affiliation":[{"name":"University of Maryland Institute for Bioscience and Biotechnology Research, Rockville, MD 20850, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matthew C.","family":"Metcalf","sequence":"additional","affiliation":[{"name":"University of Maryland Institute for Bioscience and Biotechnology Research, Rockville, MD 20850, USA"},{"name":"Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tagide","family":"deCarvalho","sequence":"additional","affiliation":[{"name":"Keith R. Porter Imaging Facility, College of Natural and Mathematical Sciences, University of Maryland, Baltimore County (UMBC), 1000 Hilltop Circle, Baltimore, MD, 21250, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Edwin","family":"Pozharski","sequence":"additional","affiliation":[{"name":"University of Maryland Institute for Bioscience and Biotechnology Research, Rockville, MD 20850, USA"},{"name":"The Center for Biomolecular Therapeutics (CBT), University of Maryland School of Medicine, Baltimore, MD, USA 21201"},{"name":"Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD, USA 21201"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuxing","family":"Li","sequence":"additional","affiliation":[{"name":"University of Maryland Institute for Bioscience and Biotechnology Research, Rockville, MD 20850, USA"},{"name":"Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD, USA"},{"name":"The Center for Biomolecular Therapeutics (CBT), University of Maryland School of Medicine, Baltimore, MD, USA 21201"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Brian G.","family":"Pierce","sequence":"additional","affiliation":[{"name":"University of Maryland Institute for Bioscience and Biotechnology Research, Rockville, MD 20850, USA"},{"name":"Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6186-6156","authenticated-orcid":false,"given":"Alexander K.","family":"Andrianov","sequence":"additional","affiliation":[{"name":"University of Maryland Institute for Bioscience and Biotechnology Research, Rockville, MD 20850, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eric A.","family":"Toth","sequence":"additional","affiliation":[{"name":"University of Maryland Institute for Bioscience and Biotechnology Research, Rockville, MD 20850, USA"},{"name":"The Center for Biomolecular Therapeutics (CBT), University of Maryland School of Medicine, Baltimore, MD, USA 21201"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9766-9995","authenticated-orcid":false,"given":"Thomas R.","family":"Fuerst","sequence":"additional","affiliation":[{"name":"University of Maryland Institute for Bioscience and Biotechnology Research, Rockville, MD 20850, USA"},{"name":"Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026080900501631000_2026.08.05.743069v1.1","doi-asserted-by":"crossref","unstructured":"Sookhoo JRV , Schiffman Z , Ambagala A , Kobasa D , Pardee K , Babiuk S . 2024. 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