Mimotope discovery as a tool to design a vaccine against Zika and dengue viruses.

Vaccine development against dengue virus is challenging because of the antibody-dependent enhancement of infection (ADE), which causes severe disease. Consecutive infections by Zika (ZIKV) and/or dengue viruses (DENV), or vaccination can predispose to ADE. Current vaccines and vaccine candidates contain the complete envelope viral protein, with epitopes that can raise antibodies causing ADE. We used the envelope dimer epitope (EDE), which induces neutralizing antibodies that do not elicit ADE, to design a vaccine against both flaviviruses. However, EDE is a discontinuous quaternary epitope that cannot be isolated from the E protein without other epitopes. Utilizing phage display, we selected three peptides that mimic the EDE. Free mimotopes were disordered and did not elicit an immune response. After their display on adeno-associated virus (AAV) capsids (VLP), they recovered their structure and were recognized by an EDE-specific antibody. Characterization by cryo-EM and enzyme-linked immunosorbent assay confirmed the correct display of a mimotope on the surface of the AAV VLP and its recognition by the specific antibody. Immunization with the AAV VLP displaying one of the mimotopes induced antibodies that recognized ZIKV and DENV. This work provides the basis for developing a Zika and dengue virus vaccine candidate that will not induce ADE.

The 1H, 15N and 13C resonance assignments of dengue virus capsid protein with the deletion of the intrinsically disordered N-terminal region.

Dengue virus belongs to the Flaviviridae family, being responsible for an endemic arboviral disease in humans. It is an enveloped virus, whose genome is a positive-stranded RNA packaged by the capsid protein. Dengue virus capsid protein (DENVC) forms homodimers in solution organized in 4 α-helices and an intrinsically disordered N-terminal region. The N-terminal region is involved in the binding of membranous structures in host cells and in the recognition of nucleotides. Here we report the 1H, 15N and 13C resonance assignments of the DENVC with the deletion of the first 19 intrinsically disordered residues. The backbone chemical shift perturbations suggest changes in the α1 and α2 helices between full length and the truncated proteins.

On the role of surrounding regions in the fusion peptide in dengue virus infection.

Dengue virus infection depends on its fusion with the host membrane, where the binding occurs through interaction between proteins on the virus cell surface and specific viral receptors on target membranes. This process is mediated by the fusion peptide located between residues 98 and 112 (DRGWGNGCGLFGKGG) that forms a loop in domain II of dengue E glycoprotein. In this study, we evaluated the role of fusion peptide surrounding regions (88-97 and 113-123) of the Dengue 2 subtype on its interaction with the membrane and fusion activity. These sequences are important to stabilize the fusion peptide loop and increase fusion activity. Three peptides, besides the fusion peptide, were synthesized by SPPS using the Fmoc chemical approach. The first contains the fusion peptide and the C-terminal region of the loop (sequence 98-123); another contains the N-terminal region (88-112) and the larger peptide contains both regions (88-123). The peptides were able to interact with a model membrane. Differences in morphology of the monolayer promoted by the peptides were assessed by Brewster Angle Microscopy (BAM). Our data indicated that the C-terminal region of fusion peptide loop is more efficient in promoting fusion and interacting with the membrane than the N-terminal sequence, which is responsible for the electrostatic initial interaction. We propose a 2-step mechanism for the interaction of the dengue virus fusion peptide with the host membrane, where the N-terminal sequence docks electrostatically on the headgroups and then the C-terminal interacts via hydrophobic forces in the acyl chains.

Nucleotide-dependent dynamics of the Dengue NS3 helicase.

Dengue represents a substantial public health burden, particularly in low-resource countries. Non-structural protein 3 (NS3) is a multifunctional protein critical in the virus life cycle and has been identified as a promising anti-viral drug target. Despite recent crystallographic studies of the NS3 helicase domain, only subtle structural nucleotide-dependent differences have been identified, such that its coupled ATPase and helicase activities remain mechanistically unclear. Here we use molecular dynamics simulations to explore the nucleotide-dependent conformational landscape of the Dengue virus NS3 helicase and identify substantial changes in the protein flexibility during the ATP hydrolysis cycle. We relate these changes to the RNA-protein interactions and proposed translocation models for other monomeric helicases. Furthermore, we report a novel open-loop conformation with a likely escape route for Pi after hydrolysis, providing new insight into the conformational changes that underlie the ATPase activity of NS3.

Expression of soluble, glycosylated and correctly folded dengue virus NS1 protein in Pichia pastoris.

The dengue virus (DENV) non structural protein 1 (NS1) is a 46-55 kDa protein that exists as homodimer inside cells and as hexamer in the extracellular milieu. Several lines of evidence have demonstrated that the biochemical and structural properties of recombinant NS1 (rNS1) vary depending on the protein expression system used. Aiming to improve current tools for studying NS1 multiple roles, a recombinant tag-free NS1 protein was expressed and purified from P. pastoris yeast cells. The best expression condition was achieved using GS115 strain and induction for 72 h with 0.7% methanol addition every 24 h. Total secreted rNS1 reached 2.18 mg in 1 L culture and the final yield of the purified protein was 1 mg per liter of culture (recovery yield of approximately 45.9%). Size-exclusion chromatography and treatment with EndoH and PNGase indicate that it exists as an N-glycosylated homodimer. Moreover, an ELISA assay with specific DENV anti-NS1 antibody that recognizes conformational epitopes revealed that rNS1 has most of its conformational epitopes preserved. The expression of rNS1 in its native conformation is an important tool for further studies of its role in DENV pathogenesis and replication.

Expression and purification of domain III proteins from Dengue and Zika viruses.

The envelope (E) protein from Dengue and Zika viruses comprises three functional and structural domains (DI, DII, and DIII). Domain III induces most of the neutralizing antibodies and, as such, is considered as having the highest antigenic potential for the evaluation of population-level surveillance and for detecting past infections in both Dengue and Zika patients. The present study aimed to clone and express recombinant proteins of domain III from Dengue virus serotype 2 and from Zika virus in a prokaryotic system, as well as evaluate their immunogenicity and cross-reactivity. Both antigens were successfully purified and their antigenicity was assessed in mice. The antibodies elicited by domain III of Zika and Dengue virus antigens recognized specifically the native proteins in infected cells. Furthermore, the antigens showed a more specific immunogenic response than that of domain III proteins from Dengue virus. The generated recombinant proteins can be potentially used in subunit vaccines or for surveillance studies.

Protein refolding based on high hydrostatic pressure and alkaline pH: Application on a recombinant dengue virus NS1 protein.

In this study we evaluated the association of high hydrostatic pressure (HHP) and alkaline pH as a minimally denaturing condition for the solubilization of inclusion bodies (IBs) generated by recombinant proteins expressed by Escherichia coli strains. The method was successfully applied to a recombinant form of the dengue virus (DENV) non-structural protein 1 (NS1). The minimal pH for IBs solubilization at 1 bar was 12 while a pH of 10 was sufficient for solubilization at HHP: 2.4 kbar for 90 min and 0.4 kbar for 14 h 30 min. An optimal refolding condition was achieved by compression of IBs at HHP and pH 10.5 in the presence of arginine, oxidized and reduced glutathiones, providing much higher yields (up to 8-fold) than association of HHP and GdnHCl via an established protocol. The refolded NS1, 109 ± 9.5 mg/L bacterial culture was recovered mainly as monomer and dimer, corresponding up to 90% of the total protein and remaining immunologically active. The proposed conditions represent an alternative for the refolding of immunologically active recombinant proteins expressed as IBs.

Molecular Aspects of the Dengue Virus Infection Process: A Review.

BACKGROUND: At the present time, dengue is one of the most important arboviruses affecting man, becoming a serious global public health problem, especially in subtropical and tropical countries, where environmental conditions favor the development and proliferation of the mosquito Aedes aegypti. Dengue is caused by a type of flavivírus, which is an enveloped virus of spherical geometry. Nowadays, it is one of the diseases with the highest incidence in Brazil, reaching the population of all states, regardless of social class. Several papers address the molecular aspects of infection of human cell by the viruses, which are reviewed in this work. CONCLUSION: Analyzing the three-dimensional structures of the fusion peptide of dengue virus protein E, we observed that the fusion peptide presents a region rich in hydrophobic residues and a "collar" of charged, polar residues. Probably, this hydrophilic collar plays an important role in the fusion process between the dengue virus and the cell membrane. In order for this disease to cease being a serious global public health problem, we must deepen our knowledge about the fusion process between the dengue virus and the cell membrane through further experimental and, especially, computational studies to find ways to inhibit the mechanism of virus infection.

Membrane vesiculation induced by proteins of the dengue virus envelope studied by molecular dynamics simulations.

Biological membranes are continuously remodeled in the cell by specific membrane-shaping machineries to form, for example, tubes and vesicles. We examine fundamental mechanisms involved in the vesiculation processes induced by a cluster of envelope (E) and membrane (M) proteins of the dengue virus (DENV) using molecular dynamics simulations and a coarse-grained model. We show that an arrangement of three E-M heterotetramers (EM3) works as a bending unit and an ordered cluster of five such units generates a closed vesicle, reminiscent of the virus budding process. In silico mutagenesis of two charged residues of the anchor helices of the envelope proteins of DENV shows that Arg-471 and Arg-60 are fundamental to produce bending stress on the membrane. The fine-tuning between the size of the EM3 unit and its specific bending action suggests this protein unit is an important factor in determining the viral particle size.

Antibody Epitopes Identified in Critical Regions of Dengue Virus Nonstructural 1 Protein in Mouse Vaccination and Natural Human Infections.

Dengue is a global public health problem and is caused by four dengue virus (DENV) serotypes (DENV1-4). A major challenge in dengue vaccine development is that cross-reactive anti-DENV Abs can be protective or potentially increase disease via Ab-dependent enhancement. DENV nonstructural protein 1 (NS1) has long been considered a vaccine candidate as it avoids Ab-dependent enhancement. In this study, we evaluated survival to challenge in a lethal DENV vascular leak model in mice immunized with NS1 combined with aluminum and magnesium hydroxide, monophosphoryl lipid A + AddaVax, or Sigma adjuvant system+CpG DNA, compared with mice infected with a sublethal dose of DENV2 and mice immunized with OVA (negative control). We characterized Ab responses to DENV1, 2, and 3 NS1 using an Ag microarray tiled with 20-mer peptides overlapping by 15 aa and identified five regions of DENV NS1 with significant levels of Ab reactivity in the NS1 + monophosphoryl lipid A + AddaVax group. Additionally, we profiled the Ab responses to NS1 of humans naturally infected with DENV2 or DENV3 in serum samples from Nicaragua collected at acute, convalescent, and 12-mo timepoints. One region in the wing domain of NS1 was immunodominant in both mouse vaccination and human infection studies, and two regions were identified only in NS1-immunized mice; thus, vaccination can generate Abs to regions that are not targeted in natural infection and could provide additional protection against lethal DENV infection. Overall, we identified a small number of immunodominant regions, which were in functionally important locations on the DENV NS1 protein and are potential correlates of protection.

The effect of the dengue non-structural 1 protein expression over the HepG2 cell proteins in a proteomic approach.

Dengue is an important mosquito borne viral disease in the world. Dengue virus (DENV) encodes a polyprotein, which is cleaved in ten proteins, including the non-structural protein 1 (NS1). In this work, we analyzed the effect of NS1 expression in one hepatic cell line, HepG2, through a shotgun proteomic approach. Cells were transfected with pcENS1 plasmid, which encodes the DENV2 NS1 protein, or the controls pcDNA3 (negative control) and pMAXGFP (GFP, a protein unrelated to dengue). Expression of NS1 was detected by immunofluorescence, western blot and flow cytometry. We identified 14,138 peptides that mapped to 4,756 proteins in all analyzed conditions. We found 41 and 81 differentially abundant proteins when compared to cells transfected with plasmids pcDNA3 and pMAXGFP, respectively. Besides, 107 proteins were detected only in the presence of NS1. We identified clusters of proteins involved mainly in mRNA process and viral RNA replication. Down regulation expression of one protein (MARCKS), identified by the proteomic analysis, was also confirmed by real time PCR in HepG2 cells infected with DENV2. Identification of proteins modulated by the presence of NS1 may improve our understanding of its role in virus infection and pathogenesis, contributing to development of new therapies and vaccines. BIOLOGICAL SIGNIFICANCE: Dengue is an important viral disease, with epidemics in tropical and subtropical regions of the world. The disease is complex, with different manifestations, in which the liver is normally affected. The NS1 is found in infected cells associated with plasma membrane and secreted into the circulation as a soluble multimer. This protein is essential for virus viability, although its function is not elucidated. Some reports indicate that the NS1 can be used as a protective antigen for the development of a dengue vaccine, while others suggest its involvement in viral pathogenesis. In this work, we report an in-depth comprehensive proteomic profiling resulting from the presence of NS1 in HepG2 cells. These results can contribute to a better understanding of the NS1 role during infection.

Dengue virus induces apoptosis in SH-SY5Y human neuroblastoma cells.

INTRODUCTION: Dengue is a human disease caused by a virus with the same name, which is transmitted by the bite of Aedes mosquitoes. The infection has a wide range of clinical presentations ranging from asymptomatic to fatal cases, with the pediatric population being the most susceptible. According to the new classification of the disease, the neurological manifestations are considered a criterion for the diagnosis of severe dengue.  OBJECTIVE: To evaluate the possible mechanisms involved in the onset of neurological signs in a cell line of human neurons as a model of infection with dengue virus type 2 (DENV-2).  MATERIALS AND METHODS: Susceptibility and permissiveness of the SH-SY5Y line to infection by DENV-2 was analyzed, showing that the proportions of viral infection and production are similar to those of primate cells used as positive control for infection.  RESULTS: Infection induced a cytopathic effect on the neuroblastoma line characterized by apoptotic cell death process, increasing the proportion of annexin V and TUNEL positive cells and an upregulation of TNF-α. Treatment with anti-TNF-α antibody increased slightly cell survival of infected cells. The addition of exogenous TNF-α to the infected cultures enhanced cell death.  CONCLUSION: These results as a whole suggest that the upregulation of TNF-α could be part of the process that induces cell damage and death in cases of dengue encephalitis.

Structural basis of potent Zika-dengue virus antibody cross-neutralization.

Zika virus is a member of the Flavivirus genus that had not been associated with severe disease in humans until the recent outbreaks, when it was linked to microcephaly in newborns in Brazil and to Guillain-Barré syndrome in adults in French Polynesia. Zika virus is related to dengue virus, and here we report that a subset of antibodies targeting a conformational epitope isolated from patients with dengue virus also potently neutralize Zika virus. The crystal structure of two of these antibodies in complex with the envelope protein of Zika virus reveals the details of a conserved epitope, which is also the site of interaction of the envelope protein dimer with the precursor membrane (prM) protein during virus maturation. Comparison of the Zika and dengue virus immunocomplexes provides a lead for rational, epitope-focused design of a universal vaccine capable of eliciting potent cross-neutralizing antibodies to protect simultaneously against both Zika and dengue virus infections.

Production of a Recombinant Dengue Virus 2 NS5 Protein and Potential Use as a Vaccine Antigen.

Dengue fever is caused by any of the four known dengue virus serotypes (DENV1 to DENV4) that affect millions of people worldwide, causing a significant number of deaths. There are vaccines based on chimeric viruses, but they still are not in clinical use. Anti-DENV vaccine strategies based on nonstructural proteins are promising alternatives to those based on whole virus or structural proteins. The DENV nonstructural protein 5 (NS5) is the main target of anti-DENV T cell-based immune responses in humans. In this study, we purified a soluble recombinant form of DENV2 NS5 expressed in Escherichia coli at large amounts and high purity after optimization of expression conditions and purification steps. The purified DENV2 NS5 was recognized by serum from DENV1-, DENV2-, DENV3-, or DENV4-infected patients in an epitope-conformation-dependent manner. In addition, immunization of BALB/c mice with NS5 induced high levels of NS5-specific antibodies and expansion of gamma interferon- and tumor necrosis factor alpha-producing T cells. Moreover, mice immunized with purified NS5 were partially protected from lethal challenges with the DENV2 NGC strain and with a clinical isolate (JHA1). These results indicate that the recombinant NS5 protein preserves immunological determinants of the native protein and is a promising vaccine antigen capable of inducing protective immune responses.

Detection of dengue NS1 and NS3 proteins in placenta and umbilical cord in fetal and maternal death.

In Brazil, dengue is a public health problem with the occurrence of explosive epidemics. This study reports maternal and fetal deaths due to dengue and which tissues of placenta and umbilical cord were analyzed by molecular methods and immunohistochemistry. The dengue NS3 and NS1 detection revealed the viral presence in different cells from placenta and umbilical cord. In the latter, DENV-2 was detected at a viral titer of 1,02 × 10(4) amounts of viral RNA. It was shown that the DENV markers analyzed here may be an alternative approach for dengue fatal cases investigation, especially involving maternal and fetal death. J. Med. Virol. 88:1448-1452, 2016. © 2016 Wiley Periodicals, Inc.

Overlapping local and long-range RNA-RNA interactions modulate dengue virus genome cyclization and replication.

The dengue virus genome is a dynamic molecule that adopts different conformations in the infected cell. Here, using RNA folding predictions, chemical probing analysis, RNA binding assays, and functional studies, we identified new cis-acting elements present in the capsid coding sequence that facilitate cyclization of the viral RNA by hybridization with a sequence involved in a local dumbbell structure at the viral 3' untranslated region (UTR). The identified interaction differentially enhances viral replication in mosquito and mammalian cells.

The protein DIIIC-2, aggregated with a specific oligodeoxynucleotide and adjuvanted in alum, protects mice and monkeys against DENV-2.

Previously, we reported the ability of the chimeric protein DIIIC-2 (domain III of the dengue envelope protein fused to the capsid protein of dengue-2 virus), to induce immunity and protection in mice, when it is highly aggregated with a non-defined oligodeoxynucleotide (ODN) and adjuvanted in alum. In this work, three different defined ODNs were studied as aggregating agents. Our results suggest that the nature of the ODN influences the capacity of protein DIIIC-2 to activate cell-mediated immunity in mice. Consequently, the ODN 39M was selected to perform further experiments in mice and nonhuman primates. Mice receiving the preparation 39M-DIIIC-2 were solidly protected against dengue virus (DENV) challenge. Moreover, monkeys immunized with the same preparation developed neutralizing antibodies, as measured by four different neutralization tests varying the virus strains and the cell lines used. Two of the immunized monkeys were completely protected against challenge, whereas the third animal had a single day of low-titer viremia. This is the first work describing the induction of short-term protection in monkeys by a formulation that is suitable for human use combining a recombinant protein from DENV with alum.

Dengue and Chikungunya differential diagnosis in pediatric patients.

INTRODUCTION: Dengue and Chikungunya infections have similar clinical symptoms, which makes their clinical diagnosis complex. Moreover, both are transmitted by the same mosquito vectors, which results in virus co-circulation and co-infection. However, the outcome of these diseases differs: Chikungunya fever is rarely fatal but can have permanent and severe rheumatic and neurological sequelae, whereas dengue disease is potentially fatal. Thus, accurate diagnosis is critical.  OBJECTIVE: To compare presumptive diagnoses based on clinical findings with the differential diagnoses based on specific laboratory tests for each virus.  MATERIALS AND METHODS: We performed specific virological and serological tests for both dengue and Chikungunya infections on eight acute-phase blood samples collected from pediatric patients with febrile syndrome. We used RT-PCR to detect dengue and Chikungunya virus, and IgM-capture ELISA to confirm infection by dengue virus.  RESULTS: Based on clinical findings, two patients were diagnosed as probable cases of dengue or Chikungunya, and two were diagnosed as probable cases of chikungunya. Four had no presumptive diagnosis of viral infection. Laboratory tests confirmed dengue infection in two patients, Chikungunya infection in two patients, and co-infection by the two viruses in the other four patients.  CONCLUSION: Clinical findings were not sufficient to make a diagnosis in pediatric patients with febrile syndrome; specific laboratory tests were required to establish the etiologic agent of the disease.

Dengue-1 envelope protein domain III produced in Pichia pastoris: potential use for serological diagnosis.

Dengue is a major international public health concern. There is no drug to treat dengue virus infections and a vaccine is yet to be licensed. The laboratory diagnosis of dengue virus infection has been greatly improved during the last decade; therefore, the main limiting factor is the production of recombinant viral antigens on a large scale. Domain III of dengue virus envelope protein contains multiplex conformation-dependent neutralizing epitopes, making it an attractive diagnostic candidate. In this work, we have demonstrated the expression of dengue virus type 1 envelope domain III protein (EDIII-D1) in methylotrophic yeast, Pichia pastoris GS115. The recombinant secreted protein (sEDIII-D1) was purified by affinity chromatography and characterized by SDS-PAGE. Purified protein was recognized in immunoblot analysis and enzyme-linked immunosorbent assay (ELISA) with dengue-infected human serum samples. In conclusion, secreted expressions of domain III protein can be obtained in P. pastoris by methanol induction. This product has the potential to be used for the diagnosis of dengue infections.

Stereochemical features of the envelope protein Domain III of dengue virus reveals putative antigenic site in the five-fold symmetry axis.

We bring to attention a characteristic parasitic pattern present in the dengue virus: it undergoes several intensive thermodynamic variations due to host environmental changes, from a vector's digestive tract, through the human bloodstream and intracellular medium. Comparatively, among the known dengue serotypes, we evaluate the effects that these medium variations may induce to the overall structural characteristics of the Domain III of the envelope (E) protein, checking for stereochemical congruences that could lead to the identification of immunologic relevant regions. We used molecular dynamics and principal component analysis to study the protein in solution, for all four dengue serotypes, under distinct pH and temperature. We stated that, while the core of Domain III is remarkably rigid and effectively unaffected by most of the mentioned intensive variations, the loops account for major and distinguishable flexibilities. Therefore, the rigidity of the Domain III core provides a foothold that projects specifically two of these high flexible loop regions towards the inner face of the envelope pores, which are found at every five-fold symmetry axis of the icosahedron-shaped mature virus. These loops bear a remarkable low identity though with high occurrence of ionizable residues, including histidines. Such stereochemical properties can provide very particular serotype-specific electrostatic surface patterns, suggesting a viral fingerprint region, on which other specific molecules and ions can establish chemical interactions in an induced fit mechanism. We assert that the proposed regions share enough relevant features to qualify for further immunologic and pharmacologic essays, such as target peptide synthesis and phage display using dengue patients' sera.