Comparative evaluation of three assays for measurement of dengue virus neutralizing antibodies

Division of Viral Diseases, Walter Reed Army Institute of Research, Silver Spring, Maryland 20910, USA.
The American journal of tropical medicine and hygiene (Impact Factor: 2.7). 08/2008; 79(1):115-22.
Source: PubMed


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.

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Available from: Jorge Pardo, Oct 28, 2015
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    • "Furthermore, there is also a lack of an effective surrogate marker of protective immunity. The plaque reduction neutralization test (PRNT) and various adaptations of this test have been used to measure neutralizing antibody titers and infer immunity (Putnak et al., 2008; Roehrig et al., 2008). However, the presence of cross-neutralizing antibodies especially following a secondary infection with a heterologous DENV serotype or flavivirus vaccination limits the ability of PRNT to serve as a surrogate marker for humoral immunity (Endy et al., 2004). "
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    ABSTRACT: Although several vaccine candidates are presently in various phases of clinical trials, the field still lacks an effective tool to determine protective immunity. The presence of cross-neutralizing antibodies limits a serological approach to identify the etiology and distinguish lifelong from short-lived humoral protection. A recent study indicated that cross-reactive but not serotype-specific antibodies require high antibody concentration to co-ligate FcγRIIB and inhibit infection. Here, we tested if these differences could allow us to distinguish serotype-specific from cross-neutralizing antibodies. Using 30 blinded early convalescent serum samples from patients with virologically confirmed dengue, we demonstrate that neutralization in the presence of FcγR-mediated phagocytosis in THP-1 correctly identifies the DENV serotype of the infection in 93.3% of the cases compared to 76.7% with plaque reduction neutralization test. Our findings could provide a new approach for evaluating DENV neutralization and suggest that in addition to blocking specific ligand-receptor interactions for viral entry, antibodies must prevent viral uncoating during FcγR-mediated phagocytosis for complete humoral protection.
    Antiviral research 10/2012; 96(3). DOI:10.1016/j.antiviral.2012.09.018 · 3.94 Impact Factor
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    • "To determine the ability of D29 Fab-IgG to neutralize Dengue infection in vitro, PRNT was carried out on BHK cells as described previously [19]. "
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    ABSTRACT: Dengue virus (DENV) is a major mosquito-borne pathogen infecting up to 100 million people each year; so far no effective treatment or vaccines are available. Recently, highly cross-reactive and infection-enhancing pre-membrane (prM)-specific antibodies were found to dominate the anti-DENV immune response in humans, raising concern over vaccine candidates that contain native dengue prM sequences. In this study, we have isolated a broadly cross-reactive prM-specific antibody, D29, during a screen with a non-immunized human Fab-phage library against the four serotypes of DENV. The antibody is capable of restoring the infectivity of virtually non-infectious immature DENV (imDENV) in FcγR-bearing K562 cells. Remarkably, D29 also cross-reacted with a cryptic epitope on the envelope (E) protein located to the DI/DII junction as evidenced by site-directed mutagenesis. This cryptic epitope, while inaccessible to antibody binding in a native virus particle, may become exposed if E is not properly folded. These findings suggest that generation of anti-prM antibodies that enhance DENV infection may not be completely avoided even with immunization strategies employing E protein alone or subunits of E proteins.
    PLoS ONE 04/2012; 7(4):e33451. DOI:10.1371/journal.pone.0033451 · 3.23 Impact Factor
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    • "PRNT also cannot be used with many vaccine strains or clinical isolates of DENV that plaque poorly or with cell types that do not support plaque formation [26], [27]. To address some of these limitations, alternative assays for detecting DENV infection and neutralization have been developed, including microneutralization and flow cytometry [6], [7], [8]. These formats have several advantages over PRNT, including single-cell detection, non-requirement for plaque formation, and relatively rapid detection (2–5 days post-infection). "
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    ABSTRACT: The lack of reliable, high-throughput tools for characterizing anti-dengue virus (DENV) antibodies in large numbers of serum samples has been an obstacle in understanding the impact of neutralizing antibodies on disease progression and vaccine efficacy. A reporter system using pseudoinfectious DENV reporter virus particles (RVPs) was previously developed by others to facilitate the genetic manipulation and biological characterization of DENV virions. In the current study, we demonstrate the diagnostic utility of DENV RVPs for measuring neutralizing antibodies in human serum samples against all four DENV serotypes, with attention to the suitability of DENV RVPs for large-scale, long-term studies. DENV RVPs used against human sera yielded serotype-specific responses and reproducible neutralization titers that were in statistical agreement with Plaque Reduction Neutralization Test (PRNT) results. DENV RVPs were also used to measure neutralization titers against the four DENV serotypes in a panel of human sera from a clinical study of dengue patients. The high-throughput capability, stability, rapidity, and reproducibility of assays using DENV RVPs offer advantages for detecting immune responses that can be applied to large-scale clinical studies of DENV infection and vaccination.
    PLoS ONE 11/2011; 6(11):e27252. DOI:10.1371/journal.pone.0027252 · 3.23 Impact Factor
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