{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,10]],"date-time":"2026-10-10T18:00:39Z","timestamp":1791655239931,"version":"4.3.4"},"reference-count":46,"publisher":"Public Library of Science (PLoS)","issue":"8","license":[{"start":{"date-parts":[[2025,8,29]],"date-time":"2025-08-29T00:00:00Z","timestamp":1756425600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Department of Biotechnology, Government of India","award":["BIRAC\/BT\/NBM0095\/02\/18"],"award-info":[{"award-number":["BIRAC\/BT\/NBM0095\/02\/18"]}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:sec id=\"sec001\">\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>Pre-existing dengue antibodies could potentially exacerbate disease severity through antibody-dependent enhancement (ADE). Current serological assays focus on measuring neutralizing antibodies for vaccine evaluation, but don\u2019t measure sub-neutralizing antibodies that enhance infection via Fc\u03b3 receptors. Consensus on a standardized system for measuring dengue virus ADE remains elusive.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec id=\"sec002\">\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>In this study, we compared and evaluated ADE responses using two different methodologies in healthy blood donors (n\u2009=\u200912) and secondary dengue patients\u2019 (n\u2009=\u200912) samples with pre-existing IgG antibodies to dengue virus (DENV). We performed an ADE-infection assay in Fc\u03b3RIIa-expressing U937, K562, and Vero-CD32a cells. Foci-reduction neutralization test (FRNT) was performed simultaneously in Vero and Vero-CD32a cells, and reduction in neutralization titres was examined in Vero-CD32a cells.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec id=\"sec003\">\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>Out of 12 blood donors, all 9 anti-dengue IgG-positive donors demonstrated ADE through infection-enhancement assay against DENV-2 and DENV-4 serotypes in U937 and K562 cells, but not in Vero-CD32a cells. None of the anti-dengue IgG-negative donor samples exhibited ADE against DENV in all three cell lines. Fold-enhancement of DENV-2 infection was comparable in the two cell lines whereas, fold-enhancement of DENV-4 infection was significantly higher in K562 than in U937 cells. Comparable neutralizing antibody titres in Vero and Vero-CD32a cells against DENV-2 and DENV-4 serotypes suggest that donor samples did not exhibit any enhancing activity in Vero-CD32a cells. Comparable DENV-2 titres and significantly lower DENV-4 titres were obtained in Vero-CD32a than in Vero cells in secondary dengue patient samples, indicating that enhancing activity was influenced by DENV serotypes.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec id=\"sec004\">\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>In summary, infection-enhancement assay using K562 cells was superior to U937 and Vero-CD32a cells in evaluating ADE. Samples with high neutralizing activity demonstrated very low levels of infection-enhancing activity in Vero-CD32a cells. Comparison of FRNT titres in Vero and Vero-CD32a cells is not suitable for detecting ADE. Our findings suggest that infection-enhancing activities are apparent at sub-neutralizing concentrations of dengue virus antibodies in all individuals exposed to dengue virus.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1371\/journal.pone.0331320","type":"journal-article","created":{"date-parts":[[2025,8,29]],"date-time":"2025-08-29T17:29:46Z","timestamp":1756488586000},"page":"e0331320","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":10,"title":["Evaluation of methods for the measurement of antibody-dependent enhancement of dengue virus infection using different Fc\u03b3RIIa expressing cell lines"],"prefix":"10.1371","volume":"20","author":[{"given":"Shweta","family":"Chelluboina","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Darshan","family":"Kshirsagar","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gauri","family":"Panzade","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Akhilesh Chandra","family":"Mishra","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vidya","family":"Arankalle","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5980-8536","authenticated-orcid":true,"given":"Shubham","family":"Shrivastava","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2025,8,29]]},"reference":[{"issue":"7446","key":"pone.0331320.ref001","doi-asserted-by":"crossref","first-page":"504","DOI":"10.1038\/nature12060","article-title":"The global distribution and burden of dengue","volume":"496","author":"S Bhatt","year":"2013","journal-title":"Nature"},{"issue":"6","key":"pone.0331320.ref002","doi-asserted-by":"crossref","first-page":"712","DOI":"10.1016\/S1473-3099(16)00026-8","article-title":"The global burden of dengue: an analysis from the Global Burden of Disease Study 2013","volume":"16","author":"JD Stanaway","year":"2016","journal-title":"Lancet Infect Dis"},{"issue":"8","key":"pone.0331320.ref003","article-title":"Dengue burden in India: recent trends and importance of climatic parameters","volume":"6","author":"SR Mutheneni","year":"2017","journal-title":"Emerg Microbes Infect"},{"key":"pone.0331320.ref004","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":"20","key":"pone.0331320.ref005","doi-asserted-by":"crossref","first-page":"10128","DOI":"10.1128\/JVI.00792-06","article-title":"Differential enhancement of dengue virus immune complex infectivity mediated by signaling-competent and signaling-incompetent human Fcgamma RIA (CD64) or FcgammaRIIA (CD32)","volume":"80","author":"WWSI Rodrigo","year":"2006","journal-title":"J Virol"},{"key":"pone.0331320.ref006","first-page":"64","article-title":"Heterogeneity in IgG- CD16 signaling in infectious disease outcomes","author":"T Wang","year":"2022","journal-title":"Immunol Rev"},{"issue":"10","key":"pone.0331320.ref007","doi-asserted-by":"crossref","first-page":"1405","DOI":"10.1093\/infdis\/jir053","article-title":"Detection of higher levels of dengue viremia using Fc\u03b3R-expressing BHK-21 cells than Fc\u03b3R-negative cells in secondary infection but not in primary infection","volume":"203","author":"ML Moi","year":"2011","journal-title":"J Infect Dis"},{"issue":"3","key":"pone.0331320.ref008","doi-asserted-by":"crossref","first-page":"225","DOI":"10.1016\/j.jcv.2011.07.009","article-title":"Dengue virus isolation relying on antibody-dependent enhancement mechanism using Fc\u03b3R-expressing BHK cells and a monoclonal antibody with infection-enhancing capacity","volume":"52","author":"ML Moi","year":"2011","journal-title":"J Clin Virol"},{"key":"pone.0331320.ref009","doi-asserted-by":"crossref","first-page":"103","DOI":"10.1099\/vir.0.014829-0","article-title":"Involvement of the Fc gamma receptor IIA cytoplasmic domain in antibody-dependent enhancement of dengue virus infection","volume":"91","author":"ML Moi","year":"2010","journal-title":"J Gen Virol"},{"issue":"11","key":"pone.0331320.ref010","doi-asserted-by":"crossref","first-page":"5659","DOI":"10.4049\/jimmunol.1203052","article-title":"Human Fc\u03b3RII cytoplasmic domains differentially influence antibody-mediated dengue virus infection","volume":"190","author":"K Boonnak","year":"2013","journal-title":"J Immunol"},{"issue":"2","key":"pone.0331320.ref011","first-page":"458","article-title":"Within-host models unravelling the dynamics of dengue reinfections","volume":"9","author":"V Anam","year":"2024","journal-title":"Infect Dis Model"},{"issue":"5596","key":"pone.0331320.ref012","doi-asserted-by":"crossref","first-page":"739","DOI":"10.1038\/265739a0","article-title":"Antibody-enhanced dengue virus infection in primate leukocytes","volume":"265","author":"SB Halstead","year":"1977","journal-title":"Nature"},{"key":"pone.0331320.ref013","first-page":"218","article-title":"Dengue viruses and mononuclear phagocytes. II. Identity of blood and tissue leukocytes supporting in vitro infection","author":"SH Allison","year":"1977","journal-title":"J Exp Med"},{"issue":"7","key":"pone.0331320.ref014","doi-asserted-by":"crossref","first-page":"816","DOI":"10.1038\/77553","article-title":"Human skin Langerhans cells are targets of dengue virus infection","volume":"6","author":"SJ Wu","year":"2000","journal-title":"Nat Med"},{"issue":"1","key":"pone.0331320.ref015","doi-asserted-by":"crossref","first-page":"17863","DOI":"10.1038\/s41598-022-21722-2","article-title":"Temporal patterns of functional anti-dengue antibodies in dengue infected individuals with different disease outcome or infection history","volume":"12","author":"HTM Vo","year":"2022","journal-title":"Sci Rep"},{"key":"pone.0331320.ref016","doi-asserted-by":"crossref","first-page":"777672","DOI":"10.3389\/fimmu.2021.777672","article-title":"Monomeric IgA antagonizes IgG-mediated enhancement of DENV infection","volume":"12","author":"AD Wegman","year":"2021","journal-title":"Front Immunol"},{"issue":"5","key":"pone.0331320.ref017","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pone.0065231","article-title":"Dengue virus neutralization in cells expressing Fc gamma receptors","volume":"8","author":"T Chawla","year":"2013","journal-title":"PLoS One"},{"issue":"4","key":"pone.0331320.ref018","doi-asserted-by":"crossref","DOI":"10.1093\/ofid\/ofv172","article-title":"Influence of Fc\u03b3RIIa-expressing cells on the assessment of neutralizing and enhancing serum antibodies elicited by a live-attenuated tetravalent dengue vaccine","volume":"2","author":"AM Byers","year":"2015","journal-title":"Open Forum Infect Dis"},{"issue":"1","key":"pone.0331320.ref019","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1186\/s12879-017-2894-7","article-title":"Dengue virus infection-enhancement activity in neutralizing antibodies of healthy adults before dengue season as determined by using Fc\u03b3R-expressing cells","volume":"18","author":"MHP Ly","year":"2018","journal-title":"BMC Infect Dis"},{"issue":"2","key":"pone.0331320.ref020","article-title":"Dengue virus infection-enhancing activity in serum samples with neutralizing activity as determined by using Fc\u03b3R-expressing cells","volume":"6","author":"ML Moi","year":"2012","journal-title":"PLoS Negl Trop Dis"},{"key":"pone.0331320.ref021","doi-asserted-by":"crossref","first-page":"591","DOI":"10.1099\/vir.0.060384-0","article-title":"Demonstration of marmosets (Callithrix jacchus) as a non-human primate model for secondary dengue virus infection: high levels of viraemia and serotype cross-reactive antibody responses consistent with secondary infection of humans","volume":"95","author":"ML Moi","year":"2014","journal-title":"J Gen Virol"},{"issue":"3","key":"pone.0331320.ref022","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pone.0092173","article-title":"Low levels of antibody-dependent enhancement in vitro using viruses and plasma from dengue patients","volume":"9","author":"P Chaichana","year":"2014","journal-title":"PLoS One"},{"issue":"10","key":"pone.0331320.ref023","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pmed.1000171","article-title":"A prospective nested case-control study of Dengue in infants: rethinking and refining the antibody-dependent enhancement dengue hemorrhagic fever model","volume":"6","author":"DH Libraty","year":"2009","journal-title":"PLoS Med"},{"issue":"14","key":"pone.0331320.ref024","doi-asserted-by":"crossref","first-page":"6503","DOI":"10.1007\/s00253-013-5021-8","article-title":"Evaluation and analysis of dengue virus enhancing and neutralizing activities using simple high-throughput assays","volume":"97","author":"X-Q Li","year":"2013","journal-title":"Appl Microbiol Biotechnol"},{"issue":"2","key":"pone.0331320.ref025","doi-asserted-by":"crossref","first-page":"297","DOI":"10.3390\/vaccines8020297","article-title":"A simple and high-throughput ELISA-based neutralization assay for the determination of anti-flavivirus neutralizing antibodies","volume":"8","author":"JC Balingit","year":"2020","journal-title":"Vaccines (Basel)"},{"key":"pone.0331320.ref026","doi-asserted-by":"crossref","first-page":"114641","DOI":"10.1016\/j.jviromet.2022.114641","article-title":"Development of a rapid assay system for detecting antibody-dependent enhancement of dengue virus infection","volume":"311","author":"A Yamanaka","year":"2023","journal-title":"J Virol Methods"},{"key":"pone.0331320.ref027","article-title":"Is Dengvaxia a useful vaccine for dengue endemic areas?","volume":"367","author":"DJ Gubler","year":"2019","journal-title":"BMJ"},{"key":"pone.0331320.ref028","author":"BS Lopes","year":"2023","journal-title":"Characterization of the neutralizing antibody response over time induced by monovalent and tetravalent dengue vaccine formulations"},{"issue":"10","key":"pone.0331320.ref029","doi-asserted-by":"crossref","first-page":"633","DOI":"10.1038\/s41577-020-00410-0","article-title":"The role of IgG Fc receptors in antibody-dependent enhancement","volume":"20","author":"S Bournazos","year":"2020","journal-title":"Nat Rev Immunol"},{"key":"pone.0331320.ref030","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/s41182-016-0004-y","article-title":"Human antibody response to dengue virus: implications for dengue vaccine design","volume":"44","author":"ML Moi","year":"2016","journal-title":"Trop Med Health"},{"issue":"139","key":"pone.0331320.ref031","article-title":"The structural basis for serotype-specific neutralization of dengue virus by a human antibody","volume":"4","author":"EP Teoh","year":"2012","journal-title":"Sci Transl Med"},{"issue":"2","key":"pone.0331320.ref032","doi-asserted-by":"crossref","first-page":"175","DOI":"10.1016\/j.virol.2009.09.024","article-title":"Dengue virus neutralization is modulated by IgG antibody subclass and Fcgamma receptor subtype","volume":"394","author":"WWSI Rodrigo","year":"2009","journal-title":"Virology"},{"issue":"4","key":"pone.0331320.ref033","doi-asserted-by":"crossref","first-page":"516","DOI":"10.1086\/590117","article-title":"Dengue in Vietnamese infants--results of infection-enhancement assays correlate with age-related disease epidemiology, and cellular immune responses correlate with disease severity","volume":"198","author":"TNB Chau","year":"2008","journal-title":"J Infect Dis"},{"issue":"2","key":"pone.0331320.ref034","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1093\/infdis\/jiw143","article-title":"Placental transfer of dengue virus (DENV)-specific antibodies and kinetics of DENV infection-enhancing activity in Brazilian infants","volume":"214","author":"PMS Castanha","year":"2016","journal-title":"J Infect Dis"},{"issue":"1","key":"pone.0331320.ref035","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pntd.0006191","article-title":"A tetravalent virus-like particle vaccine designed to display domain III of dengue envelope proteins induces multi-serotype neutralizing antibodies in mice and macaques which confer protection against antibody dependent enhancement in AG129 mice","volume":"12","author":"V Ramasamy","year":"2018","journal-title":"PLoS Negl Trop Dis"},{"key":"pone.0331320.ref036","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"},{"issue":"10","key":"pone.0331320.ref037","doi-asserted-by":"crossref","first-page":"1617","DOI":"10.1007\/s00705-010-0741-x","article-title":"Antibody-dependent enhancement of dengue virus infection in vitro by undiluted sera from monkeys infected with heterotypic dengue virus","volume":"155","author":"M Ito","year":"2010","journal-title":"Arch Virol"},{"issue":"2","key":"pone.0331320.ref038","doi-asserted-by":"crossref","first-page":"123","DOI":"10.1089\/vim.2008.0007","article-title":"Guidelines for plaque-reduction neutralization testing of human antibodies to dengue viruses","volume":"21","author":"JT Roehrig","year":"2008","journal-title":"Viral Immunol"},{"issue":"3","key":"pone.0331320.ref039","doi-asserted-by":"crossref","first-page":"402","DOI":"10.1128\/CVI.00396-09","article-title":"Discrepancy in dengue virus neutralizing antibody titers between plaque reduction neutralizing tests with Fcgamma receptor (FcgammaR)-negative and FcgammaR-expressing BHK-21 cells","volume":"17","author":"ML Moi","year":"2010","journal-title":"Clin Vaccine Immunol"},{"issue":"28","key":"pone.0331320.ref040","doi-asserted-by":"crossref","first-page":"7852","DOI":"10.1073\/pnas.1607931113","article-title":"Human antibody responses after dengue virus infection are highly cross-reactive to Zika virus","volume":"113","author":"L Priyamvada","year":"2016","journal-title":"Proc Natl Acad Sci U S A"},{"issue":"1","key":"pone.0331320.ref041","doi-asserted-by":"crossref","first-page":"1696","DOI":"10.1038\/s41467-024-45806-x","article-title":"Prior flavivirus immunity skews the yellow fever vaccine response to cross-reactive antibodies with potential to enhance dengue virus infection","volume":"15","author":"A Santos-Peral","year":"2024","journal-title":"Nat Commun"},{"key":"pone.0331320.ref042","doi-asserted-by":"crossref","first-page":"177","DOI":"10.1016\/j.ijid.2015.02.004","article-title":"Virological confirmation of concurrent dengue virus serotypes 1 and 4 by virus isolation using Fc-gamma receptor-expressing BHK cells","volume":"33","author":"ML Moi","year":"2015","journal-title":"Int J Infect Dis"},{"issue":"1","key":"pone.0331320.ref043","doi-asserted-by":"crossref","first-page":"103","DOI":"10.1007\/s00705-013-1787-3","article-title":"Determination of antibody concentration as the main parameter in a dengue virus antibody-dependent enhancement assay using Fc\u03b3R-expressing BHK cells","volume":"159","author":"ML Moi","year":"2014","journal-title":"Arch Virol"},{"issue":"1","key":"pone.0331320.ref044","doi-asserted-by":"crossref","first-page":"382","DOI":"10.1038\/s41467-023-44330-8","article-title":"Protection against symptomatic dengue infection by neutralizing antibodies varies by infection history and infecting serotype","volume":"15","author":"S Bos","year":"2024","journal-title":"Nat Commun"},{"issue":"4","key":"pone.0331320.ref045","doi-asserted-by":"crossref","first-page":"691","DOI":"10.4269\/ajtmh.15-0199","article-title":"Association of Fc\u03b3RIIa polymorphism with clinical outcome of dengue infection: first insight from Pakistan","volume":"93","author":"SN Mohsin","year":"2015","journal-title":"Am J Trop Med Hyg"},{"issue":"11","key":"pone.0331320.ref046","doi-asserted-by":"crossref","first-page":"819","DOI":"10.1016\/j.humimm.2015.09.042","article-title":"Association of FCGR2A p.R131H and CCL2 c.-2518 A>G gene variants with thrombocytopenia in patients with dengue virus infection","volume":"76","author":"K Alagarasu","year":"2015","journal-title":"Hum Immunol"}],"container-title":["PLOS One"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0331320","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,8,29]],"date-time":"2025-08-29T17:29:57Z","timestamp":1756488597000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0331320"}},"subtitle":[],"editor":[{"given":"Victoria","family":"Pando-Robles","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2025,8,29]]},"references-count":46,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2025,8,29]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0331320","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,8,29]]}}}