Immune Response to Dengue and Zika.

Flaviviruses such as dengue (DENV), yellow fever (YFV), West Nile (WNV), and Zika (ZIKV) are human pathogens of global significance. In particular, DENV causes the most prevalent mosquito-borne viral diseases in humans, and ZIKV emerged from obscurity into the spotlight in 2016 as the etiologic agent of congenital Zika syndrome. Owing to the recent emergence of ZIKV as a global pandemic threat, the roles of the immune system during ZIKV infections are as yet unclear. In contrast, decades of DENV research implicate a dual role for the immune system in protection against and pathogenesis of DENV infection. As DENV and ZIKV are closely related, knowledge based on DENV studies has been used to prioritize investigation of ZIKV immunity and pathogenesis, and to accelerate ZIKV diagnostic, therapeutic, and vaccine design. This review discusses the following topics related to innate and adaptive immune responses to DENV and ZIKV: the interferon system as the key mechanism of host defense and viral target for immune evasion, antibody-mediated protection versus antibody-dependent enhancement, and T cell-mediated protection versus original T cell antigenic sin. Understanding the mechanisms that regulate the balance between immune-mediated protection and pathogenesis during DENV and ZIKV infections is critical toward development of safe and effective DENV and ZIKV therapeutics and vaccines.

How a broadly neutralizing antibody grapples with antigenic and conformational diversity in dengue virus.

In this issue of Cell, two studies apply powerful structural approaches to probe the modes of interaction between a broadly neutralizing antibody and a conserved epitope found on four dengue virus serotypes and Zika virus. These findings offer new insights into how a broadly neutralizing antibody surmounts antigenic and conformational variation.

Human antibody C10 neutralizes by diminishing Zika but enhancing dengue virus dynamics.

The human monoclonal antibody (HmAb) C10 potently cross-neutralizes Zika virus (ZIKV) and dengue virus. Analysis of antibody fragment (Fab) C10 interactions with ZIKV and dengue virus serotype 2 (DENV2) particles by cryoelectron microscopy (cryo-EM) and amide hydrogen/deuterium exchange mass spectrometry (HDXMS) shows that Fab C10 binding decreases overall ZIKV particle dynamics, whereas with DENV2, the same Fab causes increased dynamics. Testing of different Fab C10:DENV2 E protein molar ratios revealed that, at higher Fab ratios, especially at saturated concentrations, the Fab enhanced viral dynamics (detected by HDXMS), and observation under cryo-EM showed increased numbers of distorted particles. Our results suggest that Fab C10 stabilizes ZIKV but that with DENV2 particles, high Fab C10 occupancy promotes E protein dimer conformational changes leading to overall increased particle dynamics and distortion of the viral surface. This is the first instance of a broadly neutralizing antibody eliciting virus-specific increases in whole virus particle dynamics.

The epitope arrangement on flavivirus particles contributes to Mab C10's extraordinary neutralization breadth across Zika and dengue viruses.

The human monoclonal antibody C10 exhibits extraordinary cross-reactivity, potently neutralizing Zika virus (ZIKV) and the four serotypes of dengue virus (DENV1-DENV4). Here we describe a comparative structure-function analysis of C10 bound to the envelope (E) protein dimers of the five viruses it neutralizes. We demonstrate that the C10 Fab has high affinity for ZIKV and DENV1 but not for DENV2, DENV3, and DENV4. We further show that the C10 interaction with the latter viruses requires an E protein conformational landscape that limits binding to only one of the three independent epitopes per virion. This limited affinity is nevertheless counterbalanced by the particle's icosahedral organization, which allows two different dimers to be reached by both Fab arms of a C10 immunoglobulin. The epitopes' geometric distribution thus confers C10 its exceptional neutralization breadth. Our results highlight the importance not only of paratope/epitope complementarity but also the topological distribution for epitope-focused vaccine design.

Global and cell type-specific immunological hallmarks of severe dengue progression identified via a systems immunology approach.

Severe dengue (SD) is a major cause of morbidity and mortality. To define dengue virus (DENV) target cells and immunological hallmarks of SD progression in children's blood, we integrated two single-cell approaches capturing cellular and viral elements: virus-inclusive single-cell RNA sequencing (viscRNA-Seq 2) and targeted proteomics with secretome analysis and functional assays. Beyond myeloid cells, in natural infection, B cells harbor replicating DENV capable of infecting permissive cells. Alterations in cell type abundance, gene and protein expression and secretion as well as cell-cell communications point towards increased immune cell migration and inflammation in SD progressors. Concurrently, antigen-presenting cells from SD progressors demonstrate intact uptake yet impaired interferon response and antigen processing and presentation signatures, which are partly modulated by DENV. Increased activation, regulation and exhaustion of effector responses and expansion of HLA-DR-expressing adaptive-like NK cells also characterize SD progressors. These findings reveal DENV target cells in human blood and provide insight into SD pathogenesis beyond antibody-mediated enhancement.

Protective Zika vaccines engineered to eliminate enhancement of dengue infection via immunodominance switch.

Antibody-dependent enhancement (ADE) is an important safety concern for vaccine development against dengue virus (DENV) and its antigenically related Zika virus (ZIKV) because vaccine may prime deleterious antibodies to enhance natural infections. Cross-reactive antibodies targeting the conserved fusion loop epitope (FLE) are known as the main sources of ADE. We design ZIKV immunogens engineered to change the FLE conformation but preserve neutralizing epitopes. Single vaccination conferred sterilizing immunity against ZIKV without ADE of DENV-serotype 1-4 infections and abrogated maternal-neonatal transmission in mice. Unlike the wild-type-based vaccine inducing predominately cross-reactive ADE-prone antibodies, B cell profiling revealed that the engineered vaccines switched immunodominance to dispersed patterns without DENV enhancement. The crystal structure of the engineered immunogen showed the dimeric conformation of the envelope protein with FLE disruption. We provide vaccine candidates that will prevent both ZIKV infection and infection-/vaccination-induced DENV ADE.

Immune response to dengue virus and prospects for a vaccine.

Dengue virus (DENV) is a mosquito-borne member of the Flavivirus genus and includes four serotypes (DENV-1, DENV-2, DENV-3, and DENV-4), each of which is capable of causing dengue fever and dengue hemorrhagic fever/dengue shock syndrome. Serious disease can be seen during primary infection but is more frequent following second infection with a serotype different from that of a previous infection. Infection with wild-type DENV induces high-titered neutralizing antibody that can provide long-term immunity to the homotypic virus and can provide short-term immunity (only several months duration) to a heterotypic DENV. The high level of virus replication seen during both secondary infection with a heterotypic virus and during primary DENV infection in late infancy is a direct consequence of antibody-dependent enhancement of replication. This enhanced virus replication is mediated primarily by preexisting, nonneutralizing, or subneutralizing antibodies to the virion surface antigens that enhance access of the virion-antibody complex to FcγR-bearing cells. Vaccines will need to provide long-term protection against each of the four DENV serotypes by inducing neutralizing antibodies, and live, attenuated and various nonliving virus vaccines are in development.

Comparative Flavivirus-Host Protein Interaction Mapping Reveals Mechanisms of Dengue and Zika Virus Pathogenesis.

Mosquito-borne flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), are a growing public health concern. Systems-level analysis of how flaviviruses hijack cellular processes through virus-host protein-protein interactions (PPIs) provides information about their replication and pathogenic mechanisms. We used affinity purification-mass spectrometry (AP-MS) to compare flavivirus-host interactions for two viruses (DENV and ZIKV) in two hosts (human and mosquito). Conserved virus-host PPIs revealed that the flavivirus NS5 protein suppresses interferon stimulated genes by inhibiting recruitment of the transcription complex PAF1C and that chemical modulation of SEC61 inhibits DENV and ZIKV replication in human and mosquito cells. Finally, we identified a ZIKV-specific interaction between NS4A and ANKLE2, a gene linked to hereditary microcephaly, and showed that ZIKV NS4A causes microcephaly in Drosophila in an ANKLE2-dependent manner. Thus, comparative flavivirus-host PPI mapping provides biological insights and, when coupled with in vivo models, can be used to unravel pathogenic mechanisms.

A protective Zika virus E-dimer-based subunit vaccine engineered to abrogate antibody-dependent enhancement of dengue infection.

Infections with dengue virus (DENV) and Zika virus (ZIKV) can induce cross-reactive antibody responses. Two immunodominant epitopes-one to precursor membrane protein and one to the fusion loop epitope on envelope (E) protein-are recognized by cross-reactive antibodies1-3 that are not only poorly neutralizing, but can also promote increased viral replication and disease severity via Fcγ receptor-mediated infection of myeloid cells-a process termed antibody-dependent enhancement (ADE)1,4,5. ADE is a significant concern for both ZIKV and DENV vaccines as the induction of poorly neutralizing cross-reactive antibodies may prime an individual for ADE on natural infection. In this report, we describe the design and production of covalently stabilized ZIKV E dimers, which lack precursor membrane protein and do not expose the immunodominant fusion loop epitope. Immunization of mice with ZIKV E dimers induces dimer-specific antibodies, which protect against ZIKV challenge during pregnancy. Importantly, the ZIKV E-dimer-induced response does not cross-react with DENV or induce ADE of DENV infection.

Structures of dengue virus RNA replicase complexes.

Dengue is a mosquito-borne viral infection caused by dengue virus (DENV), a member of the flaviviruses. The DENV genome is a 5'-capped positive-sense RNA with a unique 5'-stem-loop structure (SLA), which is essential for RNA replication and 5' capping. The virus-encoded proteins NS5 and NS3 are responsible for viral genome replication, but the structural basis by which they cooperatively conduct the required tasks has remained unclear. Here, we report the cryoelectron microscopy (cryo-EM) structures of SLA-bound NS5 (PC), NS3-bound PC (PC-NS3), and an RNA-elongating NS5-NS3 complex (EC). While SLA bridges the NS5 methyltransferase and RNA-dependent RNA polymerase domains in PC, the NS3 helicase domain displaces it in elongation complex (EC). The SLA- and NS3-binding sites overlap with that of human STAT2. These structures illuminate the key steps in DENV genome replication, namely, SLA-dependent replication initiation, processive RNA elongation, and 5' capping of the nascent genomic RNA, thereby providing foundations to combat flaviviruses.

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.

Molecular Insight into Dengue Virus Pathogenesis and Its Implications for Disease Control.

Dengue virus (DENV) is a mosquito-transmitted RNA virus that infects an estimated 390 million humans each year. Here, we review recent advances in our understanding of the biology of DENV and describe knowledge gaps that have impacted the development of effective vaccines and therapeutics.

Structure-Guided Design of an Anti-dengue Antibody Directed to a Non-immunodominant Epitope.

Dengue is the most common vector-borne viral disease, causing nearly 400 million infections yearly. Currently there are no approved therapies. Antibody epitopes that elicit weak humoral responses may not be accessible by conventional B cell panning methods. To demonstrate an alternative strategy to generating a therapeutic antibody, we employed a non-immunodominant, but functionally relevant, epitope in domain III of the E protein, and engineered by structure-guided methods an antibody directed to it. The resulting antibody, Ab513, exhibits high-affinity binding to, and broadly neutralizes, multiple genotypes within all four serotypes. To assess therapeutic relevance of Ab513, activity against important human clinical features of dengue was investigated. Ab513 mitigates thrombocytopenia in a humanized mouse model, resolves vascular leakage, reduces viremia to nearly undetectable levels, and protects mice in a maternal transfer model of lethal antibody-mediated enhancement. The results demonstrate that Ab513 may reduce the public health burden from dengue.

Defining Hsp70 Subnetworks in Dengue Virus Replication Reveals Key Vulnerability in Flavivirus Infection.

Viral protein homeostasis depends entirely on the machinery of the infected cell. Accordingly, viruses can illuminate the interplay between cellular proteostasis components and their distinct substrates. Here, we define how the Hsp70 chaperone network mediates the dengue virus life cycle. Cytosolic Hsp70 isoforms are required at distinct steps of the viral cycle, including entry, RNA replication, and virion biogenesis. Hsp70 function at each step is specified by nine distinct DNAJ cofactors. Of these, DnaJB11 relocalizes to virus-induced replication complexes to promote RNA synthesis, while DnaJB6 associates with capsid protein and facilitates virion biogenesis. Importantly, an allosteric Hsp70 inhibitor, JG40, potently blocks infection of different dengue serotypes in human primary blood cells without eliciting viral resistance or exerting toxicity to the host cells. JG40 also blocks replication of other medically-important flaviviruses including yellow fever, West Nile and Japanese encephalitis viruses. Thus, targeting host Hsp70 subnetworks provides a path for broad-spectrum antivirals.

Human antibodies to the dengue virus E-dimer epitope have therapeutic activity against Zika virus infection.

The Zika virus (ZIKV) epidemic has resulted in congenital abnormalities in fetuses and neonates. Although some cross-reactive dengue virus (DENV)-specific antibodies can enhance ZIKV infection in mice, those recognizing the DENV E-dimer epitope (EDE) can neutralize ZIKV infection in cell culture. We evaluated the therapeutic activity of human monoclonal antibodies to DENV EDE for their ability to control ZIKV infection in the brains, testes, placentas, and fetuses of mice. A single dose of the EDE1-B10 antibody given 3 d after ZIKV infection protected against lethality, reduced ZIKV levels in brains and testes, and preserved sperm counts. In pregnant mice, wild-type or engineered LALA variants of EDE1-B10, which cannot engage Fcg receptors, diminished ZIKV burden in maternal and fetal tissues, and protected against fetal demise. Because neutralizing antibodies to EDE have therapeutic potential against ZIKV, in addition to their established inhibitory effects against DENV, it may be possible to develop therapies that control disease caused by both viruses.

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.

Blocking NS3-NS4B interaction inhibits dengue virus in non-human primates.

Dengue is a major health threat and the number of symptomatic infections caused by the four dengue serotypes is estimated to be 96 million1 with annually around 10,000 deaths2. However, no antiviral drugs are available for the treatment or prophylaxis of dengue. We recently described the interaction between non-structural proteins NS3 and NS4B as a promising target for the development of pan-serotype dengue virus (DENV) inhibitors3. Here we present JNJ-1802-a highly potent DENV inhibitor that blocks the NS3-NS4B interaction within the viral replication complex. JNJ-1802 exerts picomolar to low nanomolar in vitro antiviral activity, a high barrier to resistance and potent in vivo efficacy in mice against infection with any of the four DENV serotypes. Finally, we demonstrate that the small-molecule inhibitor JNJ-1802 is highly effective against viral infection with DENV-1 or DENV-2 in non-human primates. JNJ-1802 has successfully completed a phase I first-in-human clinical study in healthy volunteers and was found to be safe and well tolerated4. These findings support the further clinical development of JNJ-1802, a first-in-class antiviral agent against dengue, which is now progressing in clinical studies for the prevention and treatment of dengue.

A mosquito salivary protein-driven influx of myeloid cells facilitates flavivirus transmission.

Mosquitoes transmit many disease-relevant flaviviruses. Efficient viral transmission to mammalian hosts requires mosquito salivary factors. However, the specific salivary components facilitating viral transmission and their mechanisms of action remain largely unknown. Here, we show that a female mosquito salivary gland-specific protein, here named A. aegypti Neutrophil Recruitment Protein (AaNRP), facilitates the transmission of Zika and dengue viruses. AaNRP promotes a rapid influx of neutrophils, followed by virus-susceptible myeloid cells toward mosquito bite sites, which facilitates establishment of local infection and systemic dissemination. Mechanistically, AaNRP engages TLR1 and TLR4 of skin-resident macrophages and activates MyD88-dependent NF-κB signaling to induce the expression of neutrophil chemoattractants. Inhibition of MyD88-NF-κB signaling with the dietary phytochemical resveratrol reduces AaNRP-mediated enhancement of flavivirus transmission by mosquitoes. These findings exemplify how salivary components can aid viral transmission, and suggest a potential prophylactic target.

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.