Characterization of a potent and highly unusual minimally enhancing antibody directed against dengue virus.

Dengue virus is a major pathogen, and severe infections can lead to life-threatening dengue hemorrhagic fever. Dengue virus exists as four serotypes, and dengue hemorrhagic fever is often associated with secondary heterologous infections. Antibody-dependent enhancement (ADE) may drive higher viral loads in these secondary infections and is purported to result from antibodies that recognize dengue virus but fail to fully neutralize it. Here we characterize two antibodies, 2C8 and 3H5, that bind to the envelope protein. Antibody 3H5 is highly unusual as it not only is potently neutralizing but also promotes little if any ADE, whereas antibody 2C8 has strong capacity to promote ADE. We show that 3H5 shows resilient binding in endosomal pH conditions and neutralizes at low occupancy. Immunocomplexes of 3H5 and dengue virus do not efficiently interact with Fcγ receptors, which we propose is due to the binding mode of 3H5 and constitutes the primary mechanism of how ADE is avoided.

Germline bias dictates cross-serotype reactivity in a common dengue-virus-specific CD8+ T cell response.

Adaptive immune responses protect against infection with dengue virus (DENV), yet cross-reactivity with distinct serotypes can precipitate life-threatening clinical disease. We found that clonotypes expressing the T cell antigen receptor (TCR) ß-chain variable region 11 (TRBV11-2) were 'preferentially' activated and mobilized within immunodominant human-leukocyte-antigen-(HLA)-A*11:01-restricted CD8+ T cell populations specific for variants of the nonstructural protein epitope NS3133 that characterize the serotypes DENV1, DENV3 and DENV4. In contrast, the NS3133-DENV2-specific repertoire was largely devoid of such TCRs. Structural analysis of a representative TRBV11-2+ TCR demonstrated that cross-serotype reactivity was governed by unique interplay between the variable antigenic determinant and germline-encoded residues in the second ß-chain complementarity-determining region (CDR2ß). Extensive mutagenesis studies of three distinct TRBV11-2+ TCRs further confirmed that antigen recognition was dependent on key contacts between the serotype-defined peptide and discrete residues in the CDR2ß loop. Collectively, these data reveal an innate-like mode of epitope recognition with potential implications for the outcome of sequential exposure to heterologous DENVs.

Dengue virus sero-cross-reactivity drives antibody-dependent enhancement of infection with zika virus.

Zika virus (ZIKV) was discovered in 1947 and was thought to lead to relatively mild disease. The recent explosive outbreak of ZIKV in South America has led to widespread concern, with reports of neurological sequelae ranging from Guillain Barré syndrome to microcephaly. ZIKV infection has occurred in areas previously exposed to dengue virus (DENV), a flavivirus closely related to ZIKV. Here we investigated the serological cross-reaction between the two viruses. Plasma immune to DENV showed substantial cross-reaction to ZIKV and was able to drive antibody-dependent enhancement (ADE) of ZIKV infection. Using a panel of human monoclonal antibodies (mAbs) to DENV, we showed that most antibodies that reacted to DENV envelope protein also reacted to ZIKV. Antibodies to linear epitopes, including the immunodominant fusion-loop epitope, were able to bind ZIKV but were unable to neutralize the virus and instead promoted ADE. Our data indicate that immunity to DENV might drive greater ZIKV replication and have clear implications for disease pathogenesis and future vaccine programs for ZIKV and DENV.

A new class of highly potent, broadly neutralizing antibodies isolated from viremic patients infected with dengue virus.

Dengue is a rapidly emerging, mosquito-borne viral infection, with an estimated 400 million infections occurring annually. To gain insight into dengue immunity, we characterized 145 human monoclonal antibodies (mAbs) and identified a previously unknown epitope, the envelope dimer epitope (EDE), that bridges two envelope protein subunits that make up the 90 repeating dimers on the mature virion. The mAbs to EDE were broadly reactive across the dengue serocomplex and fully neutralized virus produced in either insect cells or primary human cells, with 50% neutralization in the low picomolar range. Our results provide a path to a subunit vaccine against dengue virus and have implications for the design and monitoring of future vaccine trials in which the induction of antibody to the EDE should be prioritized.

Heterologous prime-boost immunization induces protection against dengue virus infection in cynomolgus macaques.

IMPORTANCE: Currently licensed dengue vaccines do not induce long-term protection in children without previous exposure to dengue viruses in nature. These vaccines are based on selected attenuated strains of the four dengue serotypes and employed in combination for two or three consecutive doses. In our search for a better dengue vaccine candidate, live attenuated strains were followed by non-infectious virus-like particles or the plasmids that generate these particles upon injection into the body. This heterologous prime-boost immunization induced elevated levels of virus-specific antibodies and helped to prevent dengue virus infection in a high proportion of vaccinated macaques. In macaques that remained susceptible to dengue virus, distinct mechanisms were found to account for the immunization failures, providing a better understanding of vaccine actions. Additional studies in humans in the future may help to establish whether this combination approach represents a more effective means of preventing dengue by vaccination.

Genetic diversity of the dengue virus population in dengue fever and dengue hemorrhagic fever patients.

BACKGROUND: The error-prone replication of dengue virus (DENV) in host results in the highly diverse viral population. Together with the host factor, intra-host diversity may influence the disease severity. Therefore, it is worth investigating whether there is a correlation between intra-host genetic diversity and disease severity. OBJECTIVE: To investigate the genetic diversity in DENV for four serotypes of the dengue population from patients with dengue fever (DF) and dengue hemorrhagic fever (DHF) using next-generation sequencing (NGS) technology. METHODS: Forty RNA samples categorized into eight groups by severity and serotypes were sequenced and analyzed for genetic variation. Analysis on the hot-cold genomic regions, selection pressure and correlation between genotype and disease severity were performed in this study. RESULTS: Comparison between the NGS data of the DF and DHF specimens showed conservation between their major populations with the consensus sequences for DF and DHF sharing 99% similarity. However, the minor populations in DF and DHF were more diverse. Many genes in DF had an #NS/#S ratio higher than in DHF. Only NS4B of DENV1 DF has #NS/#S ratio higher than one. Hot regions of the DF were detected in NS3 of DENV1, DENV2 and Envelope of DENV3, whereas the hot regions of the DHF samples were detected in the small region in 3'UTR of DENV2 and DENV3. CONCLUSIONS: Various explorations of the variations of DF and DHF were performed in this study. However, we have not yet found any specific characteristics of intra-host diversity associated with disease severity.

Ivermectin Accelerates Circulating Nonstructural Protein 1 (NS1) Clearance in Adult Dengue Patients: A Combined Phase 2/3 Randomized Double-blinded Placebo Controlled Trial.

BACKGROUND: Dengue is the most significant mosquito-borne viral disease; there are no specific therapeutics. The antiparasitic drug ivermectin efficiently inhibits the replication of all 4 dengue virus serotypes in vitro. METHODS: We conducted 2 consecutive randomized, double-blind, placebo-controlled trials in adult dengue patients to evaluate safety and virological and clinical efficacies of ivermectin. After a phase 2 trial with 2 or 3 days of 1 daily dose of 400 µg/kg ivermectin, we continued with a phase 3, placebo-controlled trial with 3 days of 400 µg/kg ivermectin. RESULTS: The phase 2 trial showed a trend in reduction of plasma nonstructural protein 1 (NS1) clearance time in the 3-day ivermectin group compared with placebo. Combining phase 2 and 3 trials, 203 patients were included in the intention to treat analysis (100 and 103 patients receiving ivermectin and placebo, respectively). Dengue hemorrhagic fever occurred in 24 (24.0%) of ivermectin-treated patients and 32 (31.1%) patients receiving placebo (P = .260). The median (95% confidence interval [CI]) clearance time of NS1 antigenemia was shorter in the ivermectin group (71.5 [95% CI 59.9-84.0] hours vs 95.8 [95% CI 83.9-120.0] hours, P = .014). At discharge, 72.0% and 47.6% of patients in the ivermectin and placebo groups, respectively had undetectable plasma NS1 (P = .001). There were no differences in the viremia clearance time and incidence of adverse events between the 2 groups. CONCLUSIONS: A 3-day 1 daily dose of 400 µg/kg oral ivermectin was safe and accelerated NS1 antigenemia clearance in dengue patients. However, clinical efficacy of ivermectin was not observed at this dosage regimen.

Increased capsid oligomerization is deleterious to dengue virus particle production.

The capsid protein (C) of dengue virus is required for viral infectivity as it packages viral RNA genome into infectious particles. C exists as a homodimer that forms via hydrophobic interactions between the α2 and α4 helices of monomers. To identify C region(s) important for virus particle production, a complementation system was employed in which single-round infectious particles are generated by trans-encapsidation of a viral C-deleted genome by recombinant C expressed in mosquito cells. Mutants harbouring a complete α3 deletion, or a dual Ile65-/Trp69-to-Ala substitution in the α3 helix, exhibited reduced production of infectious virus. Unexpectedly, higher proportions of oligomeric C were detected in cells expressing both mutated forms as compared with the wild-type counterpart, indicating that the α3 helix, through its internal hydrophobic residues, may down-modulate oligomerization of C during particle formation. Compared with wild-type C, the double Ile65-/Trp69 to Ala mutations appeared to hamper viral infectivity but not C and genomic RNA incorporation into the pseudo-infectious virus particles, suggesting that increased C oligomerization may impair DENV replication at the cell entry step.

A simple approach to identify functional antibody variable genes in murine hybridoma cells that coexpress aberrant kappa light transcripts by restriction enzyme digestion.

BACKGROUND: Specific binding to target protein epitopes by a mouse monoclonal antibody (mAb) relies on its variable domains. However, the isolation of functional variable gene transcripts is sometimes hindered by co-expression of aberrant transcripts in hybridoma cells. OBJECTIVE: To develop general strategies for identifying the functional variable transcripts of both heavy (VH) and kappa light (Vκ) chains from mouse hybridomas. METHODS: VH and Vκ genes of anti-dengue hybridoma clones were PCR-amplified using set of degenerate primers covering all mouse immunoglobulin families. Vκ amplicons were additionally digested with BciVI to eliminate aberrant Vκ transcripts. The productive VH and Vκ sequences were identified by Immunogenetics (IMGT) database analysis and cloned into a dual human IgG expression vector to generate chimeric antibodies (chAbs) in mammalian cells. The reactivity of chAbs was tested by immunoblot and immunofluorescent assays. RESULTS: Among 17 tested hybridoma clones, 400 bp Vκ amplicons were obtained using eight different Vκ primers. Amplicons from productive Vκ transcripts are resistant to BciVI digestion, whereas BciVI-digested amplicons indicated aberrant Vκ transcripts. 500-bp productive VH amplicons could be obtained from all clones using a set of five VH primers. The productive VH/Vκ genes of three anti-dengue NS1 mAbs (m2G6, m1F11 and m1A4) were cloned and mouse-human chAbs were generated. The binding reactivities of the chAbs to dengue NS1 were similar to the original mAbs. CONCLUSIONS: A general protocol to identify productive/functional VH and Vκ genes was demonstrated. The method is useful for producing chAbs and genetic archiving of valuable hybridoma cell lines.

Enhanced production of infectious particles by adaptive modulation of C-prM processing and C-C interaction during propagation of dengue pseudoinfectious virus in stable CprME-expressing cells.

Dengue virus assembly involves the encapsidation of genomic RNA by the capsid protein (C) and the acquisition of an envelope comprising the premembrane (prM) and envelope (E) glycoproteins. This rapid process, lacking in detectable nucleocapsid intermediates, may impose authentic C-prM-E arrangement as a prerequisite for efficient particle assembly. A mosquito cell-based complementation system was employed in this study to investigate the possibility that expression of the three structural proteins in trans allows the efficient production of a partially C-deleted dengue virus as compared to the presence of C alone. Following the transfection of ΔC56-capped RNA transcripts into C6/36 cells transiently expressing C or CprME, the production of the single-cycle virus was comparable. Subsequent propagation in the stable CprME-expressing clone, however, supported virus adaptation leading to acquisition of the L29P and S101F (PF) dual mutations in the C protein. The triple mutant, ΔC56(PF), exhibited enhanced levels of virus replication, specific infectivity and frequent increases of intracellular C dimer, as compared with ΔC56 in the CprME-clone. The PF mutations were associated with the accumulation of truncated CprM in ΔC56(PF)-infected cells, and uncleaved CprM as well as reduced intracellular C-dimer when the dual mutations were introduced into the wild-type dengue virus genetic background. These results indicate that the PF mutations may exert a replication-enhancing effect for the triple mutant virus by relieving the interference of trans-complementing structural proteins during viral assembly and suggest that the C-prM-E arrangement may be advantageous for pseudoinfectious virus production.

Longitudinal Analysis of Antibody Cross-neutralization Following Zika Virus and Dengue Virus Infection in Asia and the Americas.

Background: The 4 dengue virus serotypes (DENV1-4) and Zika virus (ZIKV) are related mosquito-borne flaviviruses of major importance globally. While monoclonal antibodies and plasma from DENV-immune donors can neutralize or enhance ZIKV in vitro and in small-animal models, and vice versa, the extent, duration, and significance of cross-reactivity in humans remains unknown, particularly in flavivirus-endemic regions. Methods: We studied neutralizing antibodies to ZIKV and DENV1-4 in longitudinal serologic specimens collected through 3 years after infection from people in Latin America and Asia with laboratory-confirmed DENV infections. We also evaluated neutralizing antibodies to ZIKV and DENV1-4 in patients with Zika through 6 months after infection. Results: In patients with Zika, the highest neutralizing antibody titers were to ZIKV, with low-level cross-reactivity to DENV1-4 that was greater in DENV-immune individuals. We found that, in primary and secondary DENV infections, neutralizing antibody titers to ZIKV were markedly lower than to the infecting DENV and heterologous DENV serotypes. Cross-neutralization was greatest in early convalescence, then ZIKV neutralization decreased, remaining at low levels over time. Conclusions: Patterns of antibody cross-neutralization suggest that ZIKV lies outside the DENV serocomplex. Neutralizing antibody titers can distinguish ZIKV from DENV infections when all viruses are analyzed simultaneously. These findings have implications for understanding natural immunity and vaccines.

Human glucose-regulated protein 78 modulates intracellular production and secretion of nonstructural protein 1 of dengue virus.

Virus-host interactions play important roles in virus infection and host cellular response. Several viruses, including dengue virus (DENV), usurp host chaperones to support their amplification and survival in the host cell. We investigated the interaction of nonstructural protein 1 (NS1) of DENV with three endoplasmic reticulum-resident chaperones (i.e. GRP78, calnexin and calreticulin) to delineate their functional roles and potential binding sites for protein complex formation. GRP78 protein showed prominent association with DENV NS1 in virus-infected Huh7 cells as evidenced by co-localization and co-immunoprecipitation assays. Further studies on the functional interaction of GRP78 protein were performed by using siRNA-mediated gene knockdown in a DENV replicon transfection system. GRP78 knockdown significantly decreased intracellular NS1 production and delayed NS1 secretion but had no effect on viral RNA replication. Dissecting the important domain of GRP78 required for DENV NS1 interaction showed co-immunoprecipitation of DENV NS1 with a full-length and substrate-binding domain (SBD), but not an ATPase domain, of GRP78, confirming their interaction through SBD binding. Molecular dynamics simulations of DENV NS1 and human GRP78 complex revealed their potential binding sites through hydrogen and hydrophobic bonding. The majority of GRP78-binding sites were located in a ß-roll domain and connector subdomains on the DENV NS1 structure involved in hydrophobic surface formation. Taken together, our findings demonstrated the roles of human GRP78 in facilitating the intracellular production and secretion of DENV NS1 as well as predicted potential binding sites between the DENV NS1 and GRP78 complex, which could have implications in the future development of target-based antiviral drugs.

Validation of genotype imputation in Southeast Asian populations and the effect of single nucleotide polymorphism annotation on imputation outcome.

BACKGROUND: Imputation involves the inference of untyped single nucleotide polymorphisms (SNPs) in genome-wide association studies. The haplotypic reference of choice for imputation in Southeast Asian populations is unclear. Moreover, the influence of SNP annotation on imputation results has not been examined. METHODS: This study was divided into two parts. In the first part, we applied imputation to genotyped SNPs from Southeast Asian populations from the Pan-Asian SNP database. Five percent of the total SNPs were removed. The remaining SNPs were applied to imputation with IMPUTE2. The imputed outcomes were verified with the removed SNPs. We compared imputation references from Chinese and Japanese haplotypes from the HapMap phase II (HMII) and the complete set of haplotypes from the 1000 Genomes Project (1000G). The second part was imputation accuracy and yield in Thai patient dataset. Half of the autosomal SNPs was removed to create Set 1. Another dataset, Set 2, was then created where we switched which half of the SNPs were removed. Both Set 1 and Set 2 were imputed with HMII to create a complete imputed SNPs dataset. The dataset was used to validate association testing, SNPs annotation and imputation outcome. RESULTS: The accuracy was highest for all populations when using the HMII reference, but at the cost of a lower yield. Thai genotypes showed the highest accuracy over other populations in both HMII and 1000G panels, although accuracy and yield varied across chromosomes. Imputation was tested in a clinical dataset to compare accuracy in gene-related regions, and coding regions were found to have a higher accuracy and yield. CONCLUSIONS: This work provides the first evidence of imputation reference selection for Southeast Asian studies and highlights the effects of SNP locations respective to genes on imputation outcome. Researchers will need to consider the trade-off between accuracy and yield in future imputation studies.

Mass spectrometric analysis of host cell proteins interacting with dengue virus nonstructural protein 1 in dengue virus-infected HepG2 cells.

Dengue virus (DENV) infection is a leading cause of the mosquito-borne infectious diseases that affect humans worldwide. Virus-host interactions appear to play significant roles in DENV replication and the pathogenesis of DENV infection. Nonstructural protein 1 (NS1) of DENV is likely involved in these processes; however, its associations with host cell proteins in DENV infection remain unclear. In this study, we used a combination of techniques (immunoprecipitation, in-solution trypsin digestion, and LC-MS/MS) to identify the host cell proteins that interact with cell-associated NS1 in an in vitro model of DENV infection in the human hepatocyte HepG2 cell line. Thirty-six novel host cell proteins were identified as potential DENV NS1-interacting partners. A large number of these proteins had characteristic binding or catalytic activities, and were involved in cellular metabolism. Coimmunoprecipitation and colocalization assays confirmed the interactions of DENV NS1 and human NIMA-related kinase 2 (NEK2), thousand and one amino acid protein kinase 1 (TAO1), and component of oligomeric Golgi complex 1 (COG1) proteins in virus-infected cells. This study reports a novel set of DENV NS1-interacting host cell proteins in the HepG2 cell line and proposes possible roles for human NEK2, TAO1, and COG1 in DENV infection.

Microparticles provide a novel biomarker to predict severe clinical outcomes of dengue virus infection.

UNLABELLED: Shedding of microparticles (MPs) is a consequence of apoptotic cell death and cellular activation. Low levels of circulating MPs in blood help maintain homeostasis, whereas increased MP generation is linked to many pathological conditions. Herein, we investigated the role of MPs in dengue virus (DENV) infection. Infection of various susceptible cells by DENV led to apoptotic death and MP release. These MPs harbored a viral envelope protein and a nonstructural protein 1 (NS1) on their surfaces. Ex vivo analysis of clinical specimens from patients with infections of different degrees of severity at multiple time points revealed that MPs generated from erythrocytes and platelets are two major MP populations in the circulation of DENV-infected patients. Elevated levels of red blood cell-derived MPs (RMPs) directly correlated with DENV disease severity, whereas a significant decrease in platelet-derived MPs was associated with a bleeding tendency. Removal by mononuclear cells of complement-opsonized NS1-anti-NS1 immune complexes bound to erythrocytes via complement receptor type 1 triggered MP shedding in vitro, a process that could explain the increased levels of RMPs in severe dengue. These findings point to the multiple roles of MPs in dengue pathogenesis. They offer a potential novel biomarker candidate capable of differentiating dengue fever from the more serious dengue hemorrhagic fever. IMPORTANCE: Dengue is the most important mosquito-transmitted viral disease in the world. No vaccines or specific treatments are available. Rapid diagnosis and immediate treatment are the keys to achieve a positive outcome. Dengue virus (DENV) infection, like some other medical conditions, changes the level and composition of microparticles (MPs), tiny bag-like structures which are normally present at low levels in the blood of healthy individuals. This study investigated how MPs in culture and patients' blood are changed in response to DENV infection. Infection of cells led to programmed cell death and MP release. In patients' blood, the majority of MPs originated from red blood cells and platelets. Decreased platelet-derived MPs were associated with a bleeding tendency, while increased levels of red blood cell-derived MPs (RMPs) correlated with more severe disease. Importantly, the level of RMPs during the early acute phase could serve as a biomarker to identify patients with potentially severe disease who require immediate care.

High Anti-Dengue Virus Activity of the OAS Gene Family Is Associated With Increased Severity of Dengue.

Dengue is a mosquito-borne viral disease that afflicts millions of individuals worldwide every year. Infection by any of the 4 dengue virus (DENV) serotypes can result in a spectrum of disease severity. We investigated the impact of variants of interferon-regulated innate immunity genes with a potent antiviral effect on the outcome of DENV infection. We compared the effect of OAS gene family variants on 2 DENV serotypes in cell culture. While both OAS1-p42 and p46 showed antiviral activity against DENV-2, only OAS1-p42 presented anti-DENV-1 activity. Conversely, whereas both OAS3_S381 and R381 variants were able to block DENV-1 infection, the anti-DENV-2 activity observed for OAS3_S381 was largely lost for the R381 variant. By means of an allelic association study of a cohort of 740 patients with dengue, we found a protective effect of OAS3_R381 against shock (odds ratio [OR], 0.37; P < .001). This effect was due to DENV-2 infections (OR, 0.13; P = .007) but was absent for DENV-1, in accordance with the serotype-dependent OAS3 activity found in the functional study. Severe dengue has long been associated with a cytokine storm of unclear origin. This work identifies an early innate immunity process that could lead to the immune overreaction observed in severe dengue and could be triggered by a specific host genotype-pathogen genotype interaction.

A simplified positive-sense-RNA virus construction approach that enhances analysis throughput.

Here we present an approach that advances the throughput of a genetic analysis of a positive-sense RNA virus by simplifying virus construction. It enabled comprehensive dissection of a complex, multigene phenotype through rapid derivation of a large number of chimeric viruses and construction of a mutant library directly from a virus pool. The versatility of the approach described here expands the applicability of diverse genetic approaches to study these viruses.

A variant in the CD209 promoter is associated with severity of dengue disease.

Dengue fever and dengue hemorrhagic fever are mosquito-borne viral diseases. Dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN1, encoded by CD209), an attachment receptor of dengue virus, is essential for productive infection of dendritic cells. Here, we report strong association between a promoter variant of CD209, DCSIGN1-336, and risk of dengue fever compared with dengue hemorrhagic fever or population controls. The G allele of the variant DCSIGN1-336 was associated with strong protection against dengue fever in three independent cohorts from Thailand, with a carrier frequency of 4.7% in individuals with dengue fever compared with 22.4% in individuals with dengue hemorrhagic fever (odds ratio for risk of dengue hemorrhagic fever versus dengue fever: 5.84, P = 1.4 x 10(-7)) and 19.5% in controls (odds ratio for protection: 4.90, P = 2 x 10(-6)). This variant affects an Sp1-like binding site and transcriptional activity in vitro. These results indicate that CD209 has a crucial role in dengue pathogenesis, which discriminates between severe dengue fever and dengue hemorrhagic fever. This may have consequences for therapeutic and preventive strategies.