Dengue reporter virus particles for measuring neutralizing antibodies against each of the four dengue serotypes

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.     

TRAIL is a novel antiviral protein against dengue virus

Dengue fever is an important tropical illness for which there is currently no virus-specific treatment. To shed light on mechanisms involved in the cellular response to dengue virus (DV), we assessed gene expression changes, using Affymetrix GeneChips (HG-U133A), of infected primary human cells and identified changes common to all cells. The common response genes included a set of 23 genes significantly induced upon DV infection of human umbilical vein endothelial cells (HUVECs), dendritic cells (DCs), monocytes, and B cells (analysis of variance, P < 0.05). Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), one of the common response genes, was identified as a key link between type I and type II interferon response genes. We found that DV induces TRAIL expression in immune cells and HUVECs at the mRNA and protein levels. The induction of TRAIL expression by DV was found to be dependent on an intact type I interferon signaling pathway. A significant increase in DV RNA accumulation was observed in anti-TRAIL antibody-treated monocytes, B cells, and HUVECs, and, conversely, a decrease in DV RNA was seen in recombinant TRAIL-treated monocytes. Furthermore, recombinant TRAIL inhibited DV titers in DV-infected DCs by an apoptosis-independent mechanism. These data suggest that TRAIL plays an important role in the antiviral response to DV infection and is a candidate for antiviral interventions against DV.     

A tetravalent dengue vaccine based on a complex adenovirus vector provides significant protection in rhesus monkeys against all four serotypes of dengue virus

Nearly a third of the human population is at risk of infection with the four serotypes of dengue viruses, and it is estimated that more than 100 million infections occur each year. A licensed vaccine for dengue viruses has become a global health priority. A major challenge to developing a dengue vaccine is the necessity to produce fairly uniform protective immune responses to all four dengue virus serotypes. We have developed two bivalent dengue virus vaccines, using a complex adenovirus vector, by incorporating the genes expressing premembrane (prM) and envelope (E) proteins of dengue virus types 1 and 2 (dengue-1 and -2, respectively) (CAdVax-Den12) or dengue-3 and -4 (CAdVax-Den34). Rhesus macaques were vaccinated by intramuscular inoculation of a tetravalent dengue vaccine formulated by combining the two bivalent vaccine constructs. Vaccinated animals produced high-titer antibodies that neutralized all four serotypes of dengue viruses in vitro. The ability of the vaccine to induce rapid, as well as sustained, protective immune responses was examined with two separate live-virus challenges administered at 4 and 24 weeks after the final vaccination. For both of these virus challenge studies, significant protection from viremia was demonstrated for all four dengue virus serotypes in vaccinated animals. Viremia from dengue-1 and dengue-3 challenges was completely blocked, whereas viremia from dengue-2 and dengue-4 was significantly reduced, as well as delayed, compared to that of control-vaccinated animals. These results demonstrate that the tetravalent dengue vaccine formulation provides significant protection in rhesus macaques against challenge with all four dengue virus serotypes.     

Bioinformatics prediction of siRNAs as potential antiviral agents against dengue viruses

Dengue virus (DENV 1-4) represents the major emerging arthropod-borne viral infection in the world. Currently, there is neither an available vaccine nor a specific treatment. Hence, there is a need of antiviral drugs for these viral infections; we describe the prediction of short interfering RNA (siRNA) as potential therapeutic agents against the four DENV serotypes. Our strategy was to carry out a series of multiple alignments using ClustalX program to find conserved sequences among the four DENV serotype genomes to obtain a consensus sequence for siRNAs design. A highly conserved sequence among the four DENV serotypes, located in the encoding sequence for NS4B and NS5 proteins was found. A total of 2,893 complete DENV genomes were downloaded from the NCBI, and after a depuration procedure to identify identical sequences, 220 complete DENV genomes were left. They were edited to select the NS4B and NS5 sequences, which were aligned to obtain a consensus sequence. Three different servers were used for siRNA design, and the resulting siRNAs were aligned to identify the most prevalent sequences. Three siRNAs were chosen, one targeted the genome region that codifies for NS4B protein and the other two; the region for NS5 protein. Predicted secondary structure for DENV genomes was used to demonstrate that the siRNAs were able to target the viral genome forming double stranded structures, necessary to activate the RNA silencing machinery.     

Coexistence of different serotypes of dengue virus

 We formulate a non–linear system of differential equations that models the dynamics of dengue fever. This disease is produced by any of the four serotypes of dengue arbovirus. Each serotype produces permanent immunity to it, but only a certain degree of cross–immunity to heterologous serotypes. In our model we consider the relation between two serotypes. Our interest is to analyze the factors that allow the invasion and persistence of different serotypes in the human population. Analysis of the model reveals the existence of four equilibrium points, which belong to the region of biological interest. One of the equilibrium points corresponds to the disease–free state, the other three equilibria correspond to the two states where just one serotype is present, and the state where both serotypes coexist, respectively. We discuss conditions for the asymptotic stability of equilibria, supported by analytical and numerical methods. We find that coexistence of both serotypes is possible for a large range of parameters. Received: 7 July 1998 / Revised version: 12 July 2002 / Published online: 26 September 2002 Keywords or phrases: Dengue fever – Primary and secondary infections – Serotype – Coexistence – Threshold – Basic reproduction number – Persistence     

Antibody germline characterization of cross-neutralizing human IgGs against 4 serotypes of dengue virus

Dengue virus (DENV), a re-emerging virus, constitutes the largest vector-borne disease virus, with 50–100 million cases reported every year. Although DENV infection induces lifelong immunity against viruses of the same serotypes, the subsequent infection with the heterologous serotypes can cause more severe form of the disease, such as Dengue Haemorrhagic Fever (DHF) or Dengue Shock Syndrome (DSS). However, there is neither approved vaccine nor specific drugs available to treat this disease. In this study, previously developed 19 human monoclonal antibodies (HuMAbs) showing strong to moderate cross neutralizing activity were selected. Most of them (13/19) were targeted to domain II of envelop glycoprotein. To understand and clarify the recognition properties, the maturation mechanisms comprising Variable/Diversity/Joining (VDJ) recombination, Variable Heavy (VH)/Variable Light (VL) chain pairing, variability at junctional site, and somatic hypermutation (SHM) of those antibodies were studied and compared with their predecessor germline sequences. IMGT/V-QUEST database was applied to analyze the isolated VH and VL sequences. To confirm the correction of isolated VH/VL, 3 HuMAbs (1A10H7, 1B3B9, 1G7C2) was transiently expressed in HEK293T cell. All three clones of the expressed recombinant IgG (rIgG) showed the same binding and neutralizing activity as same as those from hybridomas. The data obtained in this study will elucidate the properties of those HuMAbs for further genetic modification, and its binding epitopes.     

Alternative infectious entry pathways for dengue virus serotypes into mammalian cells

The entry of two dengue virus (DENV) serotypes into Vero cells was analysed using biochemical inhibitors, dominant negative mutants of cellular proteins involved in endocytic pathways, fluorescence microscopy and infectivity determinations. By treatment with dansylcadaverine and chlorpromazine and overexpression of a dominant negative form of the Eps15 protein, a clathrin-mediated endocytosis for productive DENV-1 internalization into Vero cells was demonstrated whereas the infectious entry of DENV-2 in the same cell system was independent of clathrin. Treatment with the inhibitors nystatin and methyl-β-cyclodextrin, as well as transfection of Vero cells with dominant negative caveolin-1, had no effect on DENV-2 virus infection. It was also shown, by using the K44A mutant and the inhibitor dynasore, that dynamin was required for DENV-2 entry. Consequently, the infectious entry of DENV-2 into Vero cells occurs by a non-classical endocytic pathway independent of clathrin, caveolae and lipid rafts, but dependent on dynamin. By contrast, DENV-2 entry into A549 cells was clathrin-dependent, as previously reported in HeLa, C6/36 and BS-C-1 cells. Our results conclusively show, for the first time, a differential mode of infective entry for DENV-1 and DENV-2 into a common host cell, Vero cells, as well as alternative entry pathways for a given serotype, DENV-2, into different types of cells.     

Role of dendritic cells in antibody-dependent enhancement of dengue virus infection

Dengue viruses (DV), composed of four distinct serotypes (DV1 to DV4), cause 50 to 100 million infections annually. Durable homotypic immunity follows infection but may predispose to severe subsequent heterotypic infections, a risk conferred in part by the immune response itself. Antibody-dependent enhancement (ADE), a process best described in vitro, is epidemiologically linked to complicated DV infections, especially in Southeast Asia. Here we report for the first time the ADE phenomenon in primary human dendritic cells (DC), early targets of DV infection, and human cell lines bearing Fc receptors. We show that ADE is inversely correlated with surface expression of DC-SIGN (DC-specific intercellular adhesion molecule-3-grabbing nonintegrin) and requires Fc gamma receptor IIa (FcgammaRIIa). Mature DC exhibited ADE, whereas immature DC, expressing higher levels of DC-SIGN and similar FcgammaRIIa levels, did not undergo ADE. ADE results in increased intracellular de novo DV protein synthesis, increased viral RNA production and release, and increased infectivity of the supernatants in mature DC. Interestingly, tumor necrosis factor alpha and interleukin-6 (IL-6), but not IL-10 and gamma interferon, were released in the presence of dengue patient sera but generally only at enhancement titers, suggesting a signaling component of ADE. FcgammaRIIa inhibition with monoclonal antibodies abrogated ADE and associated downstream consequences. DV versatility in entry routes (FcgammaRIIa or DC-SIGN) in mature DC broadens target options and suggests additional ways for DC to contribute to the pathogenesis of severe DV infection. Studying the cellular targets of DV infection and their susceptibility to ADE will aid our understanding of complex disease and contribute to the field of vaccine development.     

A bispecific antibody effectively neutralizes all four serotypes of dengue virus by simultaneous blocking virus attachment and fusion

Although dengue virus (DENV) infection severely threatens the health of humans, no specific antiviral drugs are currently approved for clinical use against DENV infection. Attachment and fusion are 2 critical steps for the flavivirus infection, and the corresponding functional epitopes are located at E protein domain III (E-DIII) and domain II (E-DII), respectively. Here, we constructed a bispecific antibody (DVD-1A1D-2A10) based on the 2 well-characterized anti-DENV monoclonal antibodies 1A1D-2 (1A1D) and 2A10G6 (2A10). The 1A1D antibody binds E-DIII and can block the virus attaching to the cell surface, while the 2A10 antibody binds E-DII and is able to prevent the virus from fusing with the endosomal membrane. Our data showed that DVD-1A1D-2A10 retained the antigen-binding activity of both parental antibodies. Importantly, it was demonstrated to be significantly more effective at neutralizing DENV than its parental antibodies both in vitro and in vivo, even better than the combination of them. To eliminate the potential antibody-dependent enhancement (ADE) effect, this bispecific antibody was successfully engineered to prevent Fc-γ-R interaction. Overall, we generated a bispecific anti-DENV antibody targeting both attachment and fusion stages, and this bispecific antibody broadly neutralized all 4 serotypes of DENV without risk of ADE, suggesting that it has great potential as a novel antiviral strategy against DENV.     

Gene expression analysis in human monocytes, monocyte-derived dendritic cells, and alpha-galactosylceramide-pulsed monocyte-derived dendritic cells.

In vitro proliferation and functional activation of V alpha 24NKT cells following stimulation with alpha-galactosylceramide (alpha-GalCer)-pulsed dendritic cells (DCs) have been observed. Because little is known about the molecular events on DCs following interaction with alpha-GalCer, we performed gene expression profiling of 2400 genes in monocytes and monocyte-derived immature DCs pulsed with alpha-GalCer (alpha-GalCer-imDCs). Overall, the expression levels of 48 genes were up-regulated and 28 were down-regulated in alpha-GalCer-imDCs. Semiquantitative RT-PCR analysis on monocytes, imDCs, alpha-GalCer-imDCs, and mature DCs confirmed the up- and down-regulation of the mRNA expression levels of 28 selected genes. Notably, we identified the specific up-regulation of mRNA expression levels of ribonuclease A and collapsin response mediator protein upon the stimulation of imDC with alpha-GalCer, suggesting a novel immunomodulating effect of alpha-GalCer on imDCs. In this study, we used imDCs prepared by culturing of monocytes with GM-CSF and IL-4 for 5 days and mDCs prepared by further culturing of imDCs with TNF alpha for two extra days.     

Quantitative expression and virus transmission analysis of DC-SIGN on monocyte-derived dendritic cells

The C-type lectins DC-SIGN and DC-SIGNR efficiently bind human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) strains and can transmit bound virus to adjacent CD4-positive cells. DC-SIGN also binds efficiently to the Ebola virus glycoprotein, enhancing Ebola virus infection. DC-SIGN is thought to be responsible for the ability of dendritic cells (DCs) to capture HIV and transmit it to T cells, thus promoting HIV dissemination in vitro and perhaps in vivo as well. To investigate DC-SIGN function and expression levels on DCs, we characterized a panel of monoclonal antibodies (MAbs) directed against the carbohydrate recognition domain of DC-SIGN. Using quantitative fluorescence-activated cell sorter technology, we found that DC-SIGN is highly expressed on immature monocyte-derived DCs, with at least 100,000 copies and often in excess of 250,000 copies per DC. There was modest variation (three- to fourfold) in DC-SIGN expression levels between individuals and between DCs isolated from the same individual at different times. Several MAbs efficiently blocked virus binding to cell lines expressing human or rhesus DC-SIGN, preventing HIV and SIV transmission. Interactions with Ebola virus pseudotypes were also blocked efficiently. Despite their ability to block virus-DC-SIGN interactions on cell lines, these antibodies only inhibited transmission of virus from DCs by approximately 50% or less. These results indicate that factors other than DC-SIGN may play important roles in the ability of DCs to capture and transmit HIV.     

Induction of tetravalent protective immunity against four dengue serotypes by the tandem domain III of the envelope protein

In the present study, the domain IIIs of all four dengue virus (DENV) serotypes were connected sequentially to construct the tandem domain III. The resulting DNA fragment was then cloned into pBAD/Topo ThioFusion plasmid. After induction, the Escherichia coli expression protein was purified and used to immunize BALB/c mice by subcutaneous route. The sera from mice immunized with the purified protein were confirmed to contain specific high antibody titers against DEN1, DEN2, and DEN4, and moderate antibody titer against DEN3. In suckling mouse model, 70% of the mice challenged with DEN1, DEN2, and DEN4 in combination with sera from mice immunized with the purified protein were protected, and 18% of the mice challenged with DEN3 in combination with the same sera were protected. Our data suggest that the tandem domain III of the envelope protein can be used as a potential tetravalent dengue vaccine based on a single antigen.     

Chemical structure and antiviral activity of carrageenans from Meristiella gelidium against herpes simplex and dengue virus

Chemical and spectroscopic methods showed that the major KCl-precipitated galactans from Meristiella gelidium (Solieriaceae) are iota/kappa/nu-hybrid carrageenans with the former one in higher proportion. These carrageenans showed, by HPSEC-MALLS analysis, unimodal symmetrical peaks with MW of 425.6–956.7 kDa. The effectiveness of the crude extracts from M. gelidium against HSV-2 was higher than the corresponding extract from G. griffithsiae, previously determined. However, when considering the homogeneous carrageenans, the fractions obtained from both seaweeds showed the same level of activity. The extracts and carrageenan derived from M. gelidium were more effective inhibitors of DENV-2 if compared with G. griffithsiae samples and reference polysaccharides. The most active fraction obtained from M. gelidium showed a selectivity index against HSV-2 of 25,000, a value high enough to consider this carrageenan as a promising agent to be evaluated for the treatment of genital HSV-2 infections.     

Antiviral activity of an N-allyl acridone against dengue virus

Background

Dengue virus (DENV), a member of the family Flaviviridae, is at present the most widespread causative agent of a human viral disease transmitted by mosquitoes. Despite the increasing incidence of this pathogen, there are no antiviral drugs or vaccines currently available for treatment or prevention. In a previous screening assay, we identified a group of N-allyl acridones as effective virus inhibitors. Here, the antiviral activity and mode of action targeted to viral RNA replication of one of the most active DENV-2 inhibitors was further characterized.

Results

The compound 10-allyl-7-chloro-9(10H)-acridone, designated 3b, was active to inhibit the in vitro infection of Vero cells with the four DENV serotypes, with effective concentration 50% (EC₅₀) values in the range 12.5-27.1 μM, as determined by virus yield inhibition assays. The compound was also effective in human HeLa cells. No cytotoxicity was detected at 3b concentrations up to 1000 μM. Mechanistic studies demonstrated that virus entry into the host cell was not affected, whereas viral RNA synthesis was strongly inhibited, as quantified by real time RT-PCR. The addition of exogenous guanosine together with 3b rescued only partially the infectivity of DENV-2.

Conclusions

The acridone derivative 3b selectively inhibits the infection of Vero cells with the four DENV serotypes without a direct interaction with the host cell or the virion but interfering specifically with the intracellular virus multiplication. The mode of antiviral action for this acridone apparently involves the cellular enzyme inosine-monophospahe dehydrogenase together with another still unidentified target related to DENV RNA synthesis.     

Differential effects of dengue virus on infected and bystander dendritic cells

Dendritic cells (DCs) play a central role as major targets of dengue virus (DV) infections and initiators of antiviral immune responses. Previous observations showed that DCs are activated by infection, presumably acquiring the capacity to promote cell-mediated immunity. However, separate evaluations of the maturation profiles of infected and uninfected bystander cells show that infection impairs the ability of DCs to upregulate cell surface expression of costimulatory, maturation, and major histocompatibility complex molecules, resulting in reduced T-cell stimulatory capacity. Infected DCs failed to respond to tumor necrosis factor alpha as an additional maturation stimulus and were apoptotic. Interleukin 10 (IL-10) was detected in supernatants from cultures of DV-infected DCs and cocultures of DCs and T cells. Taken together, these results constitute an immune evasion strategy used by DV that directly impairs antigen-presenting cell function by maturation blockade and induction of apoptosis.     

A heterocyclic molecule with significant activity against dengue virus

There are no specific approved drugs or vaccines for the treatment or prevention of infectious dengue virus and there are very few compounds known that inhibit the replication of this virus. This letter describes the concise synthesis of two uracil-based multifunctional compounds. One of these compounds (1) has strong activity against dengue virus. It also exhibits low activity against a few other RNA viruses, but is highly active against yellow fever virus, a related flavivirus. It is likely that the mechanism of action of the antiviral activity of this compound is through its inhibition of the enzyme, inosine monophosphate dehydrogenase (IMPDH). Molecular modeling studies reveal that the compound can have specific hydrogen bonding interactions with a number of amino acids in the active site of IMPDH, a stacking interaction with the bound natural substrate, IMP, and the ability to interfere with the binding of NAD⁺ with IMPDH, prior to the hydration step.     

Characterization of monocyte-derived IFNalpha-generated dendritic cells.

The antitumor effects of IFNalpha is mainly mediated by the activation of cytotoxic T lymphocytes (CTLs), the activation of natural killer (NK) cells, and the generation of highly potent antigen-presenting dendritic cells (IFN-DCs). Recently, we demonstrated that these cells partially express the NK cell marker CD56 and reveal a direct cytotoxic immunity towards tumor cells. The aim of the present study was to explore these cells in more detail with respect to their phenotypical and functional characteristics. Flowcytometric analyses revealed that a 5-day incubation time of CD14+ monocytes with IFNalpha results in a steady increase of CD56 surface expression of these cells from 25% (+/-2%) on day 1 up to 68% (+/-11%) on day 5. Interestingly, additional culturing of negatively selected CD56- IFN-DCs also resulted in a partial CD56 surface expression. By comparing both cell types in more detail we found a significant decrease of CD14 expression on CD56+ IFN-DCs (66+/-6%) compared to CD56- IFN-DCs (76+/-6%). On the basis of functional tests, CD56+ IFN-DCs revealed a slightly increased phagocytosis capacity compared to CD56- IFN-DCs as only 82% of CD56- IFN-DCs showed a positive intracytoplasmatic signal after 60 minutes coculturing with FITC-labeled albumin, whereas 91% of CD56+ IFN-DCs were positive. Moreover, CD56+ IFN-DCs revealed a stronger T cell stimulation capacity compared to CD56- IFN-DCs. These results together with our previously described data suggest that CD56+ IFN-DCs and CD56- IFN-DCs may represent one identical cell population with different maturation status rather than two separate cell entities. Because of their high stimulating capacity and their direct cytolytic effects these cells represent a new promising tool for cellular anticancer therapy.     

Immature monocyte-derived dendritic cells are productively infected with herpes simplex virus type 1

Herpes simplex viruses (HSV) have developed several immunoevasive strategies. Here we demonstrate a novel mechanism by which HSV type 1 may interfere with the immune response through infection of immature dendritic cells (DC) and selective downmodulation of costimulatory molecules. In our study we show productive infection of immature monocyte-derived DC, which closely resemble sessile Langerhans cells, by sequential expression of immediate-early, early, and late viral proteins and of glycoprotein D mRNA, as well as production of infectious virus of moderate titers. Infection was cytopathic, with the progressive loss of 20 to 45% of cells from 24 to 48 h after infection, with no more than 80% of DC found to be infected. These results are in contrast to those of previous findings of nonpermissive or abortive infection of monocytes and mature monocyte-derived DC. Infection of immature DC also led to selective and asynchronous downregulation of CD1a, CD40, CD54 (ICAM-1) (12 h postinfection), CD80 (24 h postinfection), and CD86 (48 h postinfection) but not of CD11c or major histocompatibility complex class I and II molecules when compared to DC exposed to UV-inactivated virus. Thus, we propose that productive infection of epidermal Langerhans cells in vivo may lead to delayed activation of T cells, allowing more time for replication of HSV type 1 in epidermal cells.