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Plaque reduction neutralization tests (PRNTs) are commonly used for measuring levels of dengue virus (DENV) neutralizing antibodies. However, these assays lack a standardized format, generally have a low sample throughput, and are labor-intensive. The objective of the present study was to evaluate two alternative DENV neutralizing antibody assays: an enzyme-linked immunosorbent assay–based microneutralization (MN) assay, and a fluorescent antibody cell sorter–based, DC-SIGN expresser dendritic cell (DC) assay. False-positive rates, serotype specificity, reproducibility, sensitivity, and agreement among the assay methods were assessed using well-characterized but limited numbers of coded test sera. Results showed that all three assays had false-positive rates of less than 10% with titers near the cut-off and generally below the estimated limits of detection. All three methods demonstrated a high degree of specificity and good agreement when used to assay sera and serum mixtures from monovalent vaccinees and sera from patients after primary natural infection, with the only notable exception being moderate-to-high neutralizing antibody titers against DENV 2 measured by PRNT in a mixture containing only DENV 3 and DENV 4 sera. The MN and DC assays demonstrated good reproducibility. All three assays were comparable in their sensitivity, except that the PRNT was less sensitive for measuring DENV 4 antibody, and the MN and DC assays were less sensitive for measuring DENV 2 antibody. However, when used to test sera from persons after tetravalent DENV vaccination or secondary DENV infection, there was poor specificity and poor agreement among the different assays.
Received August 27, 2007. Accepted for publication April 27, 2008.
Acknowledgments: We thank Dr. Mammen Mammen for providing sera from persons with naturally acquired dengue infections; and Michelle Eustache, Tsehay Aberra, and David Barvir for performing the PRNTs.
Financial support: This study was partially supported by the U.S. Military Infectious Disease Research Program. Dengue vaccine trials were jointly supported by the U.S. Army Medical Research and Materiel Command and GlaxoSmithKline Biologicals. Samples from the Queen Sirikit National Institute of Child Health were collected as part of a collaborative study between the Armed Forces Research Institute of Medical Sciences, University of Massachusetts Medical School, and Queen Sirikit National Institute of Child Health, which was supported by the U.S. Army Military Infectious Diseases Research Program and the National Institutes of Health (P01 AI34533).
Disclaimer: This material has been reviewed by the Walter Reed Army Institute of Research and GlaxoSmithKline. There is no objection to its presentation and/or publication. The opinions or assertions contained herein are the private views of the author, and are not to be construed as official, or as reflecting true views of the Department of the Army or the Department of Defense.
Disclosure: The Walter Reed Army Institute of Research and Glaxo-SmithKline have a Cooperative Research and Development Agreement for development of a dengue vaccine. J. Robert Putnak is currently conducting research sponsored by GlaxoSmithKline. This statement is made in the interest of full disclosure and not because the author considers this to be a conflict of interest.
* Address correspondence to J. Robert Putnak, Division of Viral Diseases, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Suite 3A12, Silver Spring, MD 20910. E-mail: robert.putnak{at}na.amedd.army.mil
Authors’ addresses: J. Robert Putnak and Stephen J. Thomas, Division of Viral Diseases, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Suite 3A12, Silver Spring, MD 20910, E-mails: robert.putnak{at}na.amedd.army.mil and stephen.thomas{at}na.amedd.army.mil. Rafael de la Barrera and Kenneth H. Eckels, Pilot Bio-Production Facility, Division of Regulated Activities, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910, E-mails: rafael.delabarrera{at}na.amedd.army.mil and kenneth.eckels{at}amedd.army.mil. Timothy Burgess, Viral Diseases Program, Naval Medical Research Unit 2, Jakarta; FPO, AP 96520, E-mail: burgess.namru2{at}yahoo.com. Jorge Pardo, BD Bioscience, 2350 Qume Drive, San Jose, CA 95131, E-mail: Jorge_Pardo{at}bd.com. Francis Dessy, Dirk Gheysen, and Yvers Lobet, Global Vaccine Development, GlaxoSmithKline Biologicals, 89 Rue de l’Institut, 1330 Rixensart, Belgium, E-mails: francis.dessy{at}gskbio.com, dirk.gheysen{at}gskbio.com, and yves.lobet{at}gskbio.com. Sharone Green, Center for Infectious Disease and Vaccine Research, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, E-mail: sharone.green{at}umassmed.edu. Timothy P. Endy, Department of Medicine, 1247 Weiskotten Hall, Syracuse, NY 13210, E-mail: endyt{at}upstate.edu. Bruce L. Innis, Clinical R&D and Medical Affairs, Vaccines for Virus Diseases, North America GlaxoSmithKline, 2301 Renaissance Boulevard, RN0220, PO Box 61540 King of Prussia, PA, E-mail: bruce.2.innis{at}gsk.com. Wellington Sun, Dengue Branch, Division of Vector-borne Infectious Diseases, Centers for Disease Control and Prevention, 1324 Calle Cañada, San Juan, PR 00920, E-mail: wks4{at}cdc.gov.
Reprint requests: J. Robert Putnak, Division of Viral Diseases, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Suite 3A12, Silver Spring, MD 20910, E-mail: robert.putnak{at}na.amedd.army.mil.
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