{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T01:10:37Z","timestamp":1760145037996,"version":"build-2065373602"},"reference-count":27,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2024,6,12]],"date-time":"2024-06-12T00:00:00Z","timestamp":1718150400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003569","name":"Ministry of Food and Drug Safety","doi-asserted-by":"publisher","award":["21172MFDS179"],"award-info":[{"award-number":["21172MFDS179"]}],"id":[{"id":"10.13039\/501100003569","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Recently, respiratory syncytial virus (RSV) vaccines based on the prefusion F (pre-F) antigen were approved in the United States. We aimed to develop an enzyme-linked immunosorbent assay (ELISA)-based protocol for the practical and large-scale evaluation of RSV vaccines. Two modified pre-F proteins (DS-Cav1 and SC-TM) were produced by genetic recombination and replication using an adenoviral vector. The protocol was established by optimizing the concentrations of the coating antigen (pre-F proteins), secondary antibodies, and blocking buffer. To validate the protocol, we examined its accuracy, precision, and specificity using serum samples from 150 participants across various age groups and the standard serum provided by the National Institute of Health. In the linear correlation analysis, coating concentrations of 5 and 2.5 \u03bcg\/mL of DS-Cav1 and SC-TM showed high coefficients of determination (r &gt; 0.90), respectively. Concentrations of secondary antibodies (alkaline phosphatase-conjugated anti-human immunoglobulin G, diluted 1:2000) and blocking reagents (5% skim milk\/PBS-T) were optimized to minimize non-specific reactions. High accuracy was observed for DS-Cav1 (r = 0.90) and SC-TM (r = 0.86). Further, both antigens showed high precision (coefficient of variation &lt; 15%). Inhibition ELISA revealed cross-reactivity of antibodies against DS-Cav1 and SC-TM, but not with the attachment (G) protein.<\/jats:p>","DOI":"10.3390\/v16060952","type":"journal-article","created":{"date-parts":[[2024,6,14]],"date-time":"2024-06-14T10:42:34Z","timestamp":1718361754000},"page":"952","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Development and Validation of an Enzyme-Linked Immunosorbent Assay-Based Protocol for Evaluation of Respiratory Syncytial Virus Vaccines"],"prefix":"10.3390","volume":"16","author":[{"given":"Eliel","family":"Nham","sequence":"first","affiliation":[{"name":"Division of Infectious Diseases, Department of Internal Medicine, Korea University College of Medicine, Seoul 02841, Republic of Korea"},{"name":"Vaccine Innovation Center-KU Medicine (VIC-K), Seoul 02841, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4050-2107","authenticated-orcid":false,"given":"A-Yeung","family":"Jang","sequence":"additional","affiliation":[{"name":"Division of Infectious Diseases, Department of Internal Medicine, Korea University College of Medicine, Seoul 02841, Republic of Korea"},{"name":"Vaccine Innovation Center-KU Medicine (VIC-K), Seoul 02841, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hyun Jung","family":"Ji","sequence":"additional","affiliation":[{"name":"Korea Atomic Energy Research Institute, Jeongeup 56212, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ki Bum","family":"Ahn","sequence":"additional","affiliation":[{"name":"Korea Atomic Energy Research Institute, Jeongeup 56212, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Joon-Yong","family":"Bae","sequence":"additional","affiliation":[{"name":"Vaccine Innovation Center-KU Medicine (VIC-K), Seoul 02841, Republic of Korea"},{"name":"Department of Microbiology, Korea University College of Medicine, Seoul 02841, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Man-Seong","family":"Park","sequence":"additional","affiliation":[{"name":"Vaccine Innovation Center-KU Medicine (VIC-K), Seoul 02841, Republic of Korea"},{"name":"Department of Microbiology, Korea University College of Medicine, Seoul 02841, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jin Gu","family":"Yoon","sequence":"additional","affiliation":[{"name":"Division of Infectious Diseases, Department of Internal Medicine, Korea University College of Medicine, Seoul 02841, Republic of Korea"},{"name":"Vaccine Innovation Center-KU Medicine (VIC-K), Seoul 02841, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hye","family":"Seong","sequence":"additional","affiliation":[{"name":"Division of Infectious Diseases, Department of Internal Medicine, Korea University College of Medicine, Seoul 02841, Republic of Korea"},{"name":"Vaccine Innovation Center-KU Medicine (VIC-K), Seoul 02841, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ji Yun","family":"Noh","sequence":"additional","affiliation":[{"name":"Division of Infectious Diseases, Department of Internal Medicine, Korea University College of Medicine, Seoul 02841, Republic of Korea"},{"name":"Vaccine Innovation Center-KU Medicine (VIC-K), Seoul 02841, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hee Jin","family":"Cheong","sequence":"additional","affiliation":[{"name":"Division of Infectious Diseases, Department of Internal Medicine, Korea University College of Medicine, Seoul 02841, Republic of Korea"},{"name":"Vaccine Innovation Center-KU Medicine (VIC-K), Seoul 02841, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4546-3880","authenticated-orcid":false,"given":"Woo Joo","family":"Kim","sequence":"additional","affiliation":[{"name":"Division of Infectious Diseases, Department of Internal Medicine, Korea University College of Medicine, Seoul 02841, Republic of Korea"},{"name":"Vaccine Innovation Center-KU Medicine (VIC-K), Seoul 02841, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7103-1344","authenticated-orcid":false,"given":"Ho Seong","family":"Seo","sequence":"additional","affiliation":[{"name":"Korea Atomic Energy Research Institute, Jeongeup 56212, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0148-7194","authenticated-orcid":false,"given":"Joon Young","family":"Song","sequence":"additional","affiliation":[{"name":"Division of Infectious Diseases, Department of Internal Medicine, Korea University College of Medicine, Seoul 02841, Republic of Korea"},{"name":"Vaccine Innovation Center-KU Medicine (VIC-K), Seoul 02841, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2024,6,12]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"946","DOI":"10.1016\/S0140-6736(17)30938-8","article-title":"Global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in 2015: A systematic review and modelling study","volume":"390","author":"Shi","year":"2017","journal-title":"Lancet"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"371","DOI":"10.1128\/CMR.13.3.371","article-title":"Respiratory syncytial virus infection in adults","volume":"13","author":"Falsey","year":"2000","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1069","DOI":"10.1093\/cid\/cit471","article-title":"High Morbidity and Mortality in Adults Hospitalized for Respiratory Syncytial Virus Infections","volume":"57","author":"Lee","year":"2013","journal-title":"Clin. Infect. Dis."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"12106","DOI":"10.1038\/s41598-020-69017-8","article-title":"Clinical characteristics and disease burden of respiratory syncytial virus infection among hospitalized adults","volume":"10","author":"Yoon","year":"2020","journal-title":"Sci. Rep."},{"key":"ref_5","unstructured":"WHO (2017, January 01). RSV Vaccine Research and Development Technology Roadmap. Available online: https:\/\/www.who.int\/publications\/i\/item\/WHO-IVB-17.12."},{"key":"ref_6","unstructured":"PATH (2023, June 17). RSV Vaccine and mAb Snapshot. Available online: https:\/\/www.path.org\/resources\/rsv-vaccine-and-mab-snapshot\/."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"36","DOI":"10.1016\/j.anai.2020.03.017","article-title":"The journey to a respiratory syncytial virus vaccine","volume":"125","author":"Mejias","year":"2020","journal-title":"Ann. Allergy Asthma Immunol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"9619","DOI":"10.1073\/pnas.1106536108","article-title":"Structural basis for immunization with postfusion respiratory syncytial virus fusion F glycoprotein (RSV F) to elicit high neutralizing antibody titers","volume":"108","author":"Swanson","year":"2011","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"592","DOI":"10.1126\/science.1243283","article-title":"Structure-Based Design of a Fusion Glycoprotein Vaccine for Respiratory Syncytial Virus","volume":"342","author":"McLellan","year":"2013","journal-title":"Science"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"8143","DOI":"10.1038\/ncomms9143","article-title":"A highly stable prefusion RSV F vaccine derived from structural analysis of the fusion mechanism","volume":"6","author":"Krarup","year":"2015","journal-title":"Nat. Commun."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"e0164789","DOI":"10.1371\/journal.pone.0164789","article-title":"Stability Characterization of a Vaccine Antigen Based on the Respiratory Syncytial Virus Fusion Glycoprotein","volume":"11","author":"Flynn","year":"2016","journal-title":"PLoS ONE"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"8193","DOI":"10.1128\/JVI.00133-15","article-title":"Nonglycosylated G-Protein Vaccine Protects against Homologous and Heterologous Respiratory Syncytial Virus (RSV) Challenge, while Glycosylated G Enhances RSV Lung Pathology and Cytokine Levels","volume":"89","author":"Fuentes","year":"2015","journal-title":"J. Virol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"srep42428","DOI":"10.1038\/srep42428","article-title":"Preclinical evaluation of bacterially produced RSV-G protein vaccine: Strong protection against RSV challenge in cotton rat model","volume":"7","author":"Fuentes","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Nguyen, T.N., Power, U.F., Robert, A., Haeuw, J.-F., Helffer, K., Perez, A., Asin, M.-A., Corvaia, N., and Libon, C. (2012). The Respiratory Syncytial Virus G Protein Conserved Domain Induces a Persistent and Protective Antibody Response in Rodents. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0034331"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"672","DOI":"10.1016\/j.jiac.2013.07.009","article-title":"Epidemiologic differences of four major respiratory viruses between children, adolescents, and adults in Korea","volume":"20","author":"Cheong","year":"2014","journal-title":"J. Infect. Chemother."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"124","DOI":"10.1016\/j.ebiom.2017.01.014","article-title":"Age-Specific Profiles of Antibody Responses against Respiratory Syncytial Virus Infection","volume":"16","author":"Jounai","year":"2017","journal-title":"EBioMedicine"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"e10903","DOI":"10.1097\/MD.0000000000010903","article-title":"Age-related prevalence of common upper respiratory pathogens, based on the application of the FilmArray Respiratory panel in a tertiary hospital in Greece","volume":"97","author":"Tsagarakis","year":"2018","journal-title":"Medicine"},{"key":"ref_18","first-page":"15021","article-title":"Respiratory syncytial virus infection in adults","volume":"366","author":"Nam","year":"2019","journal-title":"BMJ"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1282016","DOI":"10.3389\/fimmu.2023.1282016","article-title":"Non-glycosylated G protein with CpG ODN provides robust protection against respiratory syncytial virus without inducing eosinophilia","volume":"14","author":"Kawahara","year":"2023","journal-title":"Front. Immunol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"595","DOI":"10.1056\/NEJMoa2209604","article-title":"Respiratory Syncytial Virus Prefusion F Protein Vaccine in Older Adults","volume":"388","author":"Papi","year":"2023","journal-title":"N. Engl. J. Med."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1465","DOI":"10.1056\/NEJMoa2213836","article-title":"Efficacy and Safety of a Bivalent RSV Prefusion F Vaccine in Older Adults","volume":"388","author":"Walsh","year":"2023","journal-title":"N. Engl. J. Med."},{"key":"ref_22","unstructured":"Moderna (2023, January 17). Moderna Announces Mrna-1345, an Investigational Respiratory Syncytial Virus (Rsv) Vaccine, Has Met Primary Efficacy Endpoints in Phase 3 Trial in Older Adults. Available online: https:\/\/investors.modernatx.com\/news\/news-details\/2023\/Moderna-Announces-mRNA-1345-an-Investigational-Respiratory-Syncytial-Virus-RSV-Vaccine-Has-Met-Primary-Efficacy-Endpoints-in-Phase-3-Trial-in-Older-Adults\/default.aspx."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1451","DOI":"10.1056\/NEJMoa2216480","article-title":"Bivalent Prefusion F Vaccine in Pregnancy to Prevent RSV Illness in Infants","volume":"388","author":"Kampmann","year":"2023","journal-title":"N. Engl. J. Med."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Jenkins, V.A., Hoet, B., Hochrein, H., and De Moerlooze, L. (2023). The Quest for a Respiratory Syncytial Virus Vaccine for Older Adults: Thinking beyond the F Protein. Vaccines, 11.","DOI":"10.3390\/vaccines11020382"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"e258","DOI":"10.3346\/jkms.2022.37.e258","article-title":"Respiratory Syncytial Virus Outbreak Without Influenza in the Second Year of the Coronavirus Disease 2019 Pandemic: A National Sentinel Surveillance in Korea, 2021\u20132022 Season","volume":"37","author":"Kim","year":"2022","journal-title":"J. Korean Med Sci."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"2969","DOI":"10.3201\/eid2711.211565","article-title":"Resurgence of Respiratory Syncytial Virus Infections during COVID-19 Pandemic, Tokyo, Japan","volume":"27","author":"Ujiie","year":"2021","journal-title":"Emerg. Infect. Dis."},{"key":"ref_27","unstructured":"(2022). Risk of Severe Pressure on Healthcare Systems Due to RSV, Flu and COVID-19 Co-Circulation, European Centre for Disease Prevention and Control (ECDC)."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/16\/6\/952\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T14:57:42Z","timestamp":1760108262000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/16\/6\/952"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,6,12]]},"references-count":27,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2024,6]]}},"alternative-id":["v16060952"],"URL":"https:\/\/doi.org\/10.3390\/v16060952","relation":{},"ISSN":["1999-4915"],"issn-type":[{"type":"electronic","value":"1999-4915"}],"subject":[],"published":{"date-parts":[[2024,6,12]]}}}