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Immunization with NS1 has been shown to elicit antibody-mediated immune responses which protect mice against DENV infections. Here, we obtained peripheral blood mononuclear cells from human subjects with secondary dengue infections, which were used to construct a dengue immune phage library displaying single-chain variable fragments. Phage selective for DENV NS1 were obtained by biopanning. Twenty-one monoclonal antibodies (mAbs) against DENV NS1 were generated from the selected phage and characterized in detail. We found most anti-NS1 mAbs used IGHV1 heavy chain antibody genes. The mAbs were classified into strongly and weakly-reactive groups based on their binding to NS1 expressed in dengue virus 2 (DENV2)-infected cells. Antibody binding experiments with recombinant NS1 proteins revealed that the mAbs recognize conformational epitopes on the \u03b2-ladder domain (amino acid residues 178\u2013273) of DENV NS1. Epitope mapping studies on alanine-substituted NS1 proteins identified distinct but overlapping epitopes. Protruding amino acids distributed around the spaghetti loop are required for the binding of the strongly-reactive mAbs, whereas the recognition residues of the weakly-reactive mAbs are likely to be located in inaccessible sites facing toward the cell membrane. This information could guide the design of an NS1 epitope-based vaccine that targets cross-reactive conserved epitopes on cell surface-associated DENV NS1.<\/jats:p>","DOI":"10.1371\/journal.pone.0266136","type":"journal-article","created":{"date-parts":[[2022,5,26]],"date-time":"2022-05-26T17:28:11Z","timestamp":1653586091000},"page":"e0266136","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":8,"title":["Cross-reactive antibodies targeting surface-exposed non-structural protein 1 (NS1) of dengue virus-infected cells recognize epitopes on the spaghetti loop of the \u03b2-ladder domain"],"prefix":"10.1371","volume":"17","author":[{"given":"Romchat","family":"Kraivong","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Somchoke","family":"Traewachiwiphak","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Napon","family":"Nilchan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nattaya","family":"Tangthawornchaikul","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nuntaya","family":"Pornmun","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ranyikar","family":"Poraha","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kanokwan","family":"Sriruksa","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wannee","family":"Limpitikul","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0053-3045","authenticated-orcid":true,"given":"Panisadee","family":"Avirutnan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Prida","family":"Malasit","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1611-2667","authenticated-orcid":true,"given":"Chunya","family":"Puttikhunt","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2022,5,26]]},"reference":[{"key":"pone.0266136.ref001","doi-asserted-by":"crossref","first-page":"572681","DOI":"10.3389\/fcimb.2020.572681","article-title":"Antibody-Dependent Enhancement: A Challenge for Developing a Safe Dengue Vaccine","volume":"10","author":"R Shukla","year":"2020","journal-title":"Front Cell Infect Microbiol"},{"key":"pone.0266136.ref002","doi-asserted-by":"crossref","first-page":"421","DOI":"10.1016\/S0065-3527(03)60011-4","article-title":"Neutralization and antibody-dependent enhancement of dengue viruses","volume":"60","author":"SB Halstead","year":"2003","journal-title":"Adv Virus Res"},{"issue":"6365","key":"pone.0266136.ref003","doi-asserted-by":"crossref","first-page":"929","DOI":"10.1126\/science.aan6836","article-title":"Antibody-dependent enhancement of severe dengue disease in humans","volume":"358","author":"LC Katzelnick","year":"2017","journal-title":"Science"},{"issue":"9","key":"pone.0266136.ref004","article-title":"Development of Sanofi Pasteur tetravalent dengue vaccine","volume":"6","author":"B Guy","year":"2010","journal-title":"Hum Vaccin"},{"key":"pone.0266136.ref005","doi-asserted-by":"crossref","first-page":"k1765","DOI":"10.1136\/bmj.k1765","article-title":"WHO recommends additional tests for Sanofi\u2019s dengue vaccine after safety concerns","volume":"361","author":"G. 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