Dengue Virus Entry as Target for Antiviral Therapy

Dengue virus (DENV) infections are expanding worldwide and, because of the lack of a vaccine, the search for antiviral products is imperative. Four serotypes of DENV are described and they all cause a similar disease outcome. It would be interesting to develop an antiviral product that can interact with all four serotypes, prevent host cell infection and subsequent immune activation. DENV entry is thus an interesting target for antiviral therapy. DENV enters the host cell through receptor-mediated endocytosis. Several cellular receptors have been proposed, and DC-SIGN, present on dendritic cells, is considered as the most important DENV receptor until now. Because DENV entry is a target for antiviral therapy, various classes of compounds have been investigated to inhibit this process. In this paper, an overview is given of all the putative DENV receptors, and the most promising DENV entry inhibitors are discussed

Broad Antiviral Activity of Carbohydrate-Binding Agents against the Four Serotypes of Dengue Virus in Monocyte-Derived Dendritic Cells

BACKGROUND: Dendritic cells (DC), present in the skin, are the first target cells of dengue virus (DENV). Dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) is present on DC and recognizes N-glycosylation sites on the E-glycoprotein of DENV. Thus, the DC-SIGN/E-glycoprotein interaction can be considered as an important target for inhibitors of viral replication. We evaluated various carbohydrate-binding agents (CBAs) against all four described serotypes of DENV replication in Raji/DC-SIGN(+) cells and in monocyte-derived DC (MDDC). METHODOLOGY/PRINCIPAL FINDINGS: A dose-dependent anti-DENV activity of the CBAs Hippeastrum hybrid (HHA), Galanthus nivalis (GNA) and Urtica dioica (UDA), but not actinohivin (AH) was observed against all four DENV serotypes as analyzed by flow cytometry making use of anti-DENV antibodies. Remarkably, the potency of the CBAs against DENV in MDDC cultures was significantly higher (up to 100-fold) than in Raji/DC-SIGN(+) cells. Pradimicin-S (PRM-S), a small-size non-peptidic CBA, exerted antiviral activity in MDDC but not in Raji/DC-SIGN(+) cells. The CBAs act at an early step of DENV infection as they bind to the viral envelope of DENV and subsequently prevent virus attachment. Only weak antiviral activity of the CBAs was detected when administered after the virus attachment step. The CBAs were also able to completely prevent the cellular activation and differentiation process of MDDC induced upon DENV infection. CONCLUSIONS/SIGNIFICANCE: The CBAs exerted broad spectrum antiviral activity against the four DENV serotypes, laboratory-adapted viruses and low passage clinical isolates, evaluated in Raji/DC-SIGN(+) cells and in primary MDDC

Antiviral activity of HHA, GNA and UDA in MDDC and in Raji/DC-SIGN⁺ cells.

<p>MDDC (▾) or Raji/DC-SIGN⁺ cells (□) were infected with DENV-1, DENV-2, DENV-3 or DENV-4. Cells were incubated with increasing concentrations of HHA, GNA, UDA and viral infectivity was quantified by flow cytometry using 5 µg/ml anti-DENV mAb (clone 2H2). Data represent the percentage of inhibition of viral replication relative to the positive control (DENV-infected cells). Each value represents the mean ± SEM of 3 to 8 independent experiments.</p

Antiviral activity profile of various CBAs against the four serotypes of DENV.

a<p>EC₅₀: 50% effective concentration, or drug concentration required to inhibit DENV infection in Raji/DC-SIGN⁺ cells and MDDC by 50% as measured by viral antigen expression. Values are the mean ± SEM of 3 to 10 independent experiments. EC₅₀ values for each lectin and each DENV serotype was compared between Raji/DC-SIGN⁺ cells and MDDC. p-values were calculated with a <i>t</i>-test.</p

Dose-dependent antiviral activity of HHA, GNA and UDA in DENV-infected MDDC.

<p>(A) MDDC were infected with DENV-2 in the absence (−) or presence of dose-dependent concentrations of HHA. The number of DENV-2 positive cells was determined by flow cytometry using 5 µg/ml anti-DENV antibody recognizing the E-protein of DENV-2 (clone 3H5). In each plot, the number of DENV positive cells is indicated. (B) MDDC were infected with the four serotypes of DENV in the presence or absence of various concentrations of HHA, GNA and UDA. DENV infection was analyzed by flow cytometry using an anti-PrM antibody recognizing all four DENV serotypes (clone 2H2). % of infected cells compared to the positive virus control (VC) ± SEM of 4 to 12 different blood donors is shown.</p

Infection of MDDC by DENV.

<p>Monocytes isolated from PBMCs were untreated (A) or treated with 25 ng/ml IL-4 and 50 ng/ml GM-CSF (B) for 5 days prior to DENV-2 infection. Two days after infection the cells were permeabilized and analyzed for DC-SIGN expression and DENV infection by confocal microscopy and flow cytometry. Uninfected cells were stained with a PE-labeled monoclonal DC-SIGN-antibody (red). Infected cells were stained with a mixture of antibodies recognizing DENV-2 E-protein and PrM protein (green). Nuclei were stained with DAPI (blue). Infected monocytes (A) and MDDC (B) were analyzed by flow cytometry to detect DENV-2 positive cells. The values indicated in each dot plot represent the % of DENV-2 positive cells. (C) MDDC were preincubated with 10 µg/ml of isotype control IgG_2a, anti-DC-SIGN or anti-MR antibody for 30 minutes before DENV-2 infection. Viral replication was analyzed by flow cytometry. % Inhibition of viral replication ± SEM of 4 different blood donors is shown.</p

Antiviral assays with DENV-2 in Raji/DC-SIGN⁺ cells and HHA.

<p>Raji/DC-SIGN⁺ cells were infected with DENV-2 under four different experimental conditions. (•) Standard antiviral assay: cells were infected with DENV-2 together with HHA (2000-400-80-16-3.2 nM) and incubated at 37°C to allow internalization of the virus. (□) Prebinding assay: cells were pretreated with HHA (2000-400-80-16 nM) before binding to DENV-2. (▴) Postbinding assay: cells were infected with DENV-2 and after washing to remove unbound virus, HHA was added dose-dependently (2000-400-80-16 nM). (∇) Pre-exposure assay: cell-free DENV-2 was pre-exposed to HHA prior to exposure to Raji/DC-SIGN⁺ cells. Raji/DC-SIGN⁺ cells were infected with a 50-fold dilution of HHA-exposed DENV (final HHA concentration: 40-8-1.6-0.32 nM). For all four conditions, cells were washed twice to remove virus and/or compound and collected after 4 days. Viral infectivity was analyzed by flow cytometry. Data represent the mean % of inhibition of viral replication ± SEM of 3 independent experiments.</p

Differentiation process of MDDC induced by DENV-2 and inhibition of this process by CBAs.

<p>MDDC were infected with DENV-2 in the absence or presence of 400 nM HHA, GNA or 2.3 µM UDA. 5 days post infection, cell surface expression of CD80, CD86, DC-SIGN and MR were analyzed by flow cytometry with specific PE-labeled mAbs. Shown is the surface expression of the marker in uninfected cell culture (dashed line), untreated DENV-2-infected cell culture (full line) and the CBA-treated DENV-2-infected cell culture (full histogram). Data shown here are from 1 representative donor out of 4 different blood donors.</p