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Here, we used structure-based design to identify and characterize amino acid substitutions that stabilize gB in its metastable prefusion conformation. One variant containing two engineered interprotomer disulfide bonds and two cavity-filling substitutions (gB-C7), displayed increased expression and thermostability. A 2.8 \u00c5 resolution cryoelectron microscopy structure shows that gB-C7 adopts a prefusion-like conformation, revealing additional structural elements at the membrane-distal apex. Unlike previous observations for several class I viral fusion proteins, mice immunized with postfusion or prefusion-stabilized forms of soluble gB protein displayed similar neutralizing antibody titers, here specifically against an HCMV laboratory strain on fibroblasts. Collectively, these results identify initial strategies to stabilize class III viral fusion proteins and provide tools to probe gB-directed antibody responses.<\/jats:p>","DOI":"10.1073\/pnas.2404250121","type":"journal-article","created":{"date-parts":[[2024,9,4]],"date-time":"2024-09-04T13:55:39Z","timestamp":1725458139000},"update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":42,"title":["Structure-based design of a soluble human cytomegalovirus glycoprotein B antigen stabilized in a prefusion-like conformation"],"prefix":"10.1073","volume":"121","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0671-670X","authenticated-orcid":false,"given":"Madeline R.","family":"Sponholtz","sequence":"first","affiliation":[{"name":"Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 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