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This intracellular humoral response relies on opsonized viral particles reaching the cytosol intact but the antigenic and kinetic constraints involved are unknown. We have solved the structure of a potent TRIM21-dependent neutralizing antibody in complex with human adenovirus 5 hexon and show how these properties influence immune activity. Structure-guided mutagenesis was used to generate antibodies with 20,000-fold variation in affinity, on-rates that differ by ~50-fold and off-rates by &gt;175-fold. Characterization of these variants during infection revealed that TRIM21-dependent neutralization and NF\u03baB activation was largely unaffected by on-rate kinetics. In contrast, TRIM21 antiviral activity was exquisitely dependent upon off-rate, with sub-\u03bcM affinity antibodies nevertheless unable to stimulate signaling because of fast dissociation kinetics. These results define the antibody properties required to elicit an efficient intracellular immune response during viral infection.<\/jats:p>","DOI":"10.1038\/srep37457","type":"journal-article","created":{"date-parts":[[2016,11,24]],"date-time":"2016-11-24T10:05:02Z","timestamp":1479981902000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":33,"title":["Antibody-antigen kinetics constrain intracellular humoral immunity"],"prefix":"10.1038","volume":"6","author":[{"given":"Maria","family":"Bottermann","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Heidrun Elisabeth","family":"Lode","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ruth E.","family":"Watkinson","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stian","family":"Foss","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Inger","family":"Sandlie","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jan Terje","family":"Andersen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Leo C.","family":"James","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2016,11,24]]},"reference":[{"key":"BFsrep37457_CR1","doi-asserted-by":"publisher","first-page":"6200","DOI":"10.1073\/pnas.0609174104","volume":"104","author":"LC James","year":"2007","unstructured":"James, L. 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