{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,3]],"date-time":"2026-07-03T18:07:24Z","timestamp":1783102044866,"version":"3.54.6"},"update-to":[{"DOI":"10.1371\/journal.pntd.0013010","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2025,4,22]],"date-time":"2025-04-22T00:00:00Z","timestamp":1745280000000}}],"reference-count":30,"publisher":"Public Library of Science (PLoS)","issue":"4","license":[{"start":{"date-parts":[[2025,4,10]],"date-time":"2025-04-10T00:00:00Z","timestamp":1744243200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001407","name":"Department of Biotechnology, Ministry of Science and Technology, India","doi-asserted-by":"publisher","award":["BT\/INF\/22\/SP53419\/2024"],"award-info":[{"award-number":["BT\/INF\/22\/SP53419\/2024"]}],"id":[{"id":"10.13039\/501100001407","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Government of Kerala"}],"content-domain":{"domain":["www.plosntds.org"],"crossmark-restriction":false},"short-container-title":["PLoS Negl Trop Dis"],"abstract":"<jats:sec id=\"sec021\">\n<jats:title>Background<\/jats:title>\n<jats:p>Rabies is a fatal, but vaccine-preventable disease. The conventional \u2018gold standard\u2019 recommended for estimating anti-rabies vaccine immune response is Rapid Fluorescent Focus Inhibition Test (RFFIT). Here, we describe a pseudovirus-based neutralization assay (PVNT) as an alternative method to detect the neutralizing antibody response.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec022\">\n<jats:title>Methodology\/Principal Findings<\/jats:title>\n<jats:p>We used a recombinant Vesicular Stomatitis Virus (rVSV)-based pseudotyping system in HEK293 cells transfected with rabies G glycoprotein to produce high titre pseudoviruses. These were confirmed by neutralization with anti-G monoclonal antibodies and a reference serum, as well as immunofluorescence-based detection of pseudovirus infection. A secreted alkaline phosphatase (SEAP) reporter was included to make the assay high-throughput adaptable with readouts possible using a conventional ELISA reader. A PVNT was developed that had a dynamic range of OD<jats:sub>405<\/jats:sub> from 0.10 to 2.00, and was used to test a set of RFFIT-validated 71 human serum samples. Results of PVNT and RFFIT showed significant concordance with a Pearson\u2019s correlation value of 0.8555. In samples with border-line RFFIT positive values (a RFFIT titre in the range of 0.5-1.8 IU\/mL), concordance was low, with the PVNT results tending to be negative. PVNT assay had a specificity of 100% when compared to RFFIT. The assay also had 100% positive predicative value indicating its robustness for use in the evaluation for the presence of neutralizing antibodies against a near 100% fatal disease such as rabies.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec023\">\n<jats:title>Conclusions\/Significance<\/jats:title>\n<jats:p>Our results show that PVNT is a reproducible, sensitive and specific assay with an objective and documentable readout. The assay is faster with a turnaround time of less than 24h and can be automated for large scale screening. Importantly, it can be performed without the restrictions in handling live rabies virus. PVNT provides a reliable alternative to RFFIT for monitoring vaccine efficacy and immunity status, making it a valuable tool in rabies control programs.<\/jats:p>\n<\/jats:sec>","DOI":"10.1371\/journal.pntd.0013010","type":"journal-article","created":{"date-parts":[[2025,4,10]],"date-time":"2025-04-10T17:30:01Z","timestamp":1744306201000},"page":"e0013010","update-policy":"https:\/\/doi.org\/10.1371\/journal.pntd.corrections_policy","source":"Crossref","is-referenced-by-count":6,"title":["Assessing anti-rabies vaccine response in humans: A rapid and high-throughput adaptable, pseudovirus-based neutralization assay as an alternative to rapid fluorescent focus inhibition test (RFFIT)"],"prefix":"10.1371","volume":"19","author":[{"given":"Santhik S.","family":"Lupitha","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Geetu Rose","family":"Varghese","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lekshmi J.","family":"Das","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Priya","family":"Prabhakaran","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ashwini M.","family":"Ananda","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Reeta S.","family":"Mani","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6345-3590","authenticated-orcid":true,"given":"Easwaran","family":"Sreekumar","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2025,4,10]]},"reference":[{"issue":"9951","key":"pntd.0013010.ref001","doi-asserted-by":"crossref","first-page":"1389","DOI":"10.1016\/S0140-6736(13)62707-5","article-title":"Current status of rabies and prospects for elimination","volume":"384","author":"AR Fooks","year":"2014","journal-title":"Lancet"},{"issue":"4","key":"pntd.0013010.ref002","doi-asserted-by":"crossref","first-page":"e0003709","DOI":"10.1371\/journal.pntd.0003709","article-title":"Estimating the global burden of endemic canine rabies","volume":"9","author":"K Hampson","year":"2015","journal-title":"PLoS Negl Trop Dis"},{"issue":"2","key":"pntd.0013010.ref003","doi-asserted-by":"crossref","first-page":"291","DOI":"10.1016\/j.antiviral.2013.03.011","article-title":"Eliminating canine rabies, the principal source of human infection: what will it take?","volume":"98","author":"F-X Meslin","year":"2013","journal-title":"Antiviral Res"},{"key":"pntd.0013010.ref004","volume-title":"World Health Organization expert consultation on rabies: third report","author":"WHO","year":"2018"},{"key":"pntd.0013010.ref005","first-page":"201","article-title":"WHO position paper: April","volume":"16","author":"Vaccines Rabies","year":"2018","journal-title":"Wkly Epidemiol Rec"},{"issue":"1","key":"pntd.0013010.ref006","doi-asserted-by":"crossref","first-page":"451","DOI":"10.1146\/annurev-virology-100114-055157","article-title":"Everything you always wanted to know about rabies virus (but were afraid to ask)","volume":"2","author":"BM Davis","year":"2015","journal-title":"Annu Rev Virol"},{"issue":"5","key":"pntd.0013010.ref007","doi-asserted-by":"crossref","first-page":"3711","DOI":"10.1128\/JVI.72.5.3711-3719.1998","article-title":"Collaboration of antibody and inflammation in clearance of rabies virus from the central nervous system","volume":"72","author":"DC Hooper","year":"1998","journal-title":"J Virol"},{"issue":"18","key":"pntd.0013010.ref008","doi-asserted-by":"crossref","first-page":"6547","DOI":"10.1007\/s00253-021-11515-4","article-title":"Detection and quantification of anti-rabies glycoprotein antibodies: current state and perspectives","volume":"105","author":"MC Rodriguez","year":"2021","journal-title":"Appl Microbiol Biotechnol"},{"issue":"4","key":"pntd.0013010.ref009","doi-asserted-by":"crossref","first-page":"570","DOI":"10.3181\/00379727-32-7767P","article-title":"Early diagnosis of rabies by mouse inoculation. 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