Abstract
Dengue virus (DENV) is an emerging virus that threatens over two-third of the world’s population. The specific diagnosis of dengue infection by serology is based on assays that detect DENV-specific antibodies including neutralizing antibodies (Abs). Neutralizing Abs are an important, if not the main, mechanism of protection from natural dengue virus (DENV) infection as well. The current gold-standard assay for measuring neutralizing Ab responses against DENV is the plaque reduction neutralization assay (PRNT). However, this assay is slow and laborious and utilizes physiologically irrelevant cell lines. Here, we describe a relatively high-throughput, flow cytometry-based neutralization assay for DENV that has been optimized for use with a human monocytic suspension cell line, U937 + DC-SIGN, or the more commonly used adherent monkey kidney cells, Vero-81.
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References
Murphy BR, Whitehead SS (2011) Immune response to dengue virus and prospects for a vaccine. Annu Rev Immunol 29:587–619. doi:10.1146/annurev-immunol-031210-101315
Simmons CP, Farrar JJ, Nguyen v V, Wills B (2012) Dengue. N Engl J Med 366(15):1423–1432. doi:10.1056/NEJMra1110265
Moi ML, Lim CK, Kotaki A, Takasaki T, Kurane I (2010) Discrepancy in dengue virus neutralizing antibody titers between plaque reduction neutralizing tests with Fcgamma receptor (FcgammaR)-negative and FcgammaR-expressing BHK-21 cells. Clin Vaccine Immunol 17(3):402–407. doi:10.1128/CVI.00396-09
Moi ML, Lim CK, Chua KB, Takasaki T, Kurane I (2012) Dengue virus infection-enhancing activity in serum samples with neutralizing activity as determined by using FcgammaR-expressing cells. PLoS Negl Trop Dis 6(2):e1536. doi:10.1371/journal.pntd.0001536
Wu RS, Chan KR, Tan HC, Chow A, Allen JC Jr, Ooi EE (2012) Neutralization of dengue virus in the presence of Fc receptor-mediated phagocytosis distinguishes serotype-specific from cross-neutralizing antibodies. Antiviral Res 96(3):340–343. doi:10.1016/j.antiviral.2012.09.018
Wahala WM, Kraus AA, Haymore LB, Accavitti-Loper MA, de Silva AM (2009) Dengue virus neutralization by human immune sera: role of envelope protein domain III-reactive antibody. Virology 392(1):103–113. doi:10.1016/j.virol.2009.06.037
Wahala WM, Donaldson EF, de Alwis R, Accavitti-Loper MA, Baric RS, de Silva AM (2010) Natural strain variation and antibody neutralization of dengue serotype 3 viruses. PLoS Pathog 6(3):e1000821. doi:10.1371/journal.ppat.1000821
Diamond MS, Harris E, Ennis FA (2012) Defeating dengue: a challenge for a vaccine. Nat Med 18(11):1622–1623. doi:10.1038/nm.2997
Kraus AA, Messer W, Haymore LB, de Silva AM (2007) Comparison of plaque- and flow cytometry-based methods for measuring dengue virus neutralization. J Clin Microbiol 45(11):3777–3780. doi:10.1128/JCM.00827-07
Navarro-Sanchez E, Altmeyer R, Amara A, Schwartz O, Fieschi F, Virelizier JL, Arenzana-Seisdedos F, Despres P (2003) Dendritic-cell-specific ICAM3-grabbing non-integrin is essential for the productive infection of human dendritic cells by mosquito-cell-derived dengue viruses. EMBO Rep 4(7):723–728. doi:10.1038/sj.embor.embor866
Sakuntabhai A, Turbpaiboon C, Casademont I, Chuansumrit A, Lowhnoo T, Kajaste-Rudnitski A, Kalayanarooj SM, Tangnararatchakit K, Tangthawornchaikul N, Vasanawathana S, Chaiyaratana W, Yenchitsomanus PT, Suriyaphol P, Avirutnan P, Chokephaibulkit K, Matsuda F, Yoksan S, Jacob Y, Lathrop GM, Malasit P, Despres P, Julier C (2005) A variant in the CD209 promoter is associated with severity of dengue disease. Nat Genet 37(5):507–513. doi:10.1038/ng1550
Zompi S, Santich BH, Beatty PR, Harris E (2011) Protection from secondary dengue virus infection in a mouse model reveals the role of serotype cross-reactive B and T cells. J Immunol 188(1):404–416. doi: jimmunol.1102124, [pii] 10.4049/jimmunol.1102124
Yokoyama WM, Christensen M, Santos GD, Miller D (2006) Production of monoclonal antibodies. Curr Protoc Immunol Chapter 2:Unit 2 5. doi:10.1002/0471142735.im0205s74
Henchal EA, Gentry MK, McCown JM, Brandt WE (1982) Dengue virus-specific and flavivirus group determinants identified with monoclonal antibodies by indirect immunofluorescence. Am J Trop Med Hyg 31(4):830–836
Kaufman BM, Summers PL, Dubois DR, Cohen WH, Gentry MK, Timchak RL, Burke DS, Eckels KH (1989) Monoclonal antibodies for dengue virus prM glycoprotein protect mice against lethal dengue infection. Am J Trop Med Hyg 41(5):576–580
Acknowledgments
We thank Anne Broadwater for excellent technical assistance. These studies were supported by a Pediatric Dengue Vaccine Initiative Targeted Research Grant and by National Institutes of Health Grant U54 AI057157 (PI: F. Sparling) from the Southeastern Regional Center of Excellence for Emerging Infections and Biodefense.
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de Alwis, R., de Silva, A.M. (2014). Measuring Antibody Neutralization of Dengue Virus (DENV) Using a Flow Cytometry-Based Technique. In: Padmanabhan, R., Vasudevan, S. (eds) Dengue. Methods in Molecular Biology, vol 1138. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0348-1_3
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DOI: https://doi.org/10.1007/978-1-4939-0348-1_3
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