A unified route for flavivirus structures uncovers essential pocket factors conserved across pathogenic viruses.

The epidemic emergence of relatively rare and geographically isolated flaviviruses adds to the ongoing disease burden of viruses such as dengue. Structural analysis is key to understand and combat these pathogens. Here, we present a chimeric platform based on an insect-specific flavivirus for the safe and rapid structural analysis of pathogenic viruses. We use this approach to resolve the architecture of two neurotropic viruses and a structure of dengue virus at 2.5 Å, the highest resolution for an enveloped virion. These reconstructions allow improved modelling of the stem region of the envelope protein, revealing two lipid-like ligands within highly conserved pockets. We show that these sites are essential for viral growth and important for viral maturation. These findings define a hallmark of flavivirus virions and a potential target for broad-spectrum antivirals and vaccine design. We anticipate the chimeric platform to be widely applicable for investigating flavivirus biology.

Eficacia y seguridad de la vacuna contra el dengue / Efficacy and safety of dengue vaccine

El dengue es un problema creciente para la salud pública mundial. En Argentina, los casos se han ido incrementado en los últimos años. La vacuna Dengvaxia (CYD-TDV) fue aprobada por la Agencia Nacional de Medicamentos, Alimentos y Tecnología (ANMAT) en 2017, y actualmente está indicada para personas entre los 9 y 45 años de edad que residan enzonas endémicas. A partir de la consulta de una paciente sobre la posibilidad de vacunarse contra el dengue, la autora se plantea la pertinencia de su indicación, teniendo en cuenta la eficacia y seguridad de la vacuna. Luego de una búsqueda rápida se encontró evidencia que señala que la vacuna contra el dengue CYD-TDV mostró poca eficacia en comparación con otras vacunas disponibles en el mercado, siendo más segura y eficaz en personas que ya han sido infectadas anteriormente por el virus del dengue (sujetos seropositivos). En cambio, se observó un aumento del riesgo de dengue grave en los infectados por vez primera tras la vacunación (sujetos seronegativos). Se concluye que la estrategia recomendada consiste en vacunar únicamente a las personas que hayan tenido infección por dengue con anterioridad, consistiendo en una buena práctica la toma de decisiones compartidas con cada paciente. (AU)

Dengue is a growing problem for global public health. In Argentina, cases have been increasing in recent years. The Dengvaxia vaccine (CYD-TDV) was approved by the National Agency for Medicines, Food and Technology in 2017, and it is currently indicated for people between 9 and 45 years of age who reside in endemic areas. Based on the consultation of a patient about the possibility of being vaccinated against dengue, the author considers the relevance of its indication, taking into account the efficacy and safety of the vaccine. After a quick search, evidence was found that indicates that the CYD-TDV dengue vaccine showed little efficacy compared to other vaccines available on the market, being safer and more effective in people who have already been previously infected by the dengue virus (seropositive subjects). In contrast, an increased risk of severe dengue was observed in those infected for the first time after vaccination (seronegative subjects). It is concluded that the recommended strategy consists of vaccinating only people who have had dengue infection before, making shared decisions with each patient a good practice. (AU)

A Non-structural 1 Protein G53D Substitution Attenuates a Clinically Tested Live Dengue Vaccine.

The molecular basis of dengue virus (DENV) attenuation remains ambiguous and hampers a targeted approach to derive safe but nonetheless immunogenic live vaccine candidates. Here, we take advantage of DENV serotype 2 PDK53 vaccine strain, which recently and successfully completed a phase-3 clinical trial, to identify how this virus is attenuated compared to its wild-type parent, DENV2 16681. Site-directed mutagenesis on a 16681 infectious clone identifies a single G53D substitution in the non-structural 1 (NS1) protein that reduces 16681 infection and dissemination in both Aedes aegypti, as well as in mammalian cells to produce the characteristic phenotypes of PDK53. Mechanistically, NS1 G53D impairs the function of a known host factor, the endoplasmic reticulum (ER)-resident ribophorin 1 protein, to properly glycosylate NS1 and thus induce a host antiviral gene through ER stress responses. Our findings provide molecular insights on DENV attenuation on a clinically tested strain.

Amino acid starvation enhances vaccine efficacy by augmenting neutralizing antibody production.

Specific reduction in the intake of proteins or amino acids (AAs) offers enormous health benefits, including increased life span, protection against age-associated disorders, and improved metabolic fitness and immunity. Cells respond to conditions of AA starvation by activating the amino acid starvation response (AAR). Here, we showed that mimicking AAR with halofuginone (HF) enhanced the magnitude and affinity of neutralizing, antigen-specific antibody responses in mice immunized with dengue virus envelope domain III protein (DENVrEDIII), a potent vaccine candidate against DENV. HF enhanced the formation of germinal centers (GCs) and increased the production of the cytokine IL-10 in the secondary lymphoid organs of vaccinated mice. Furthermore, HF promoted the transcription of genes associated with memory B cell formation and maintenance and maturation of GCs in the draining lymph nodes of vaccinated mice. The increased abundance of IL-10 in HF-preconditioned mice correlated with enhanced GC responses and may promote the establishment of long-lived plasma cells that secrete antigen-specific, high-affinity antibodies. Thus, these data suggest that mimetics of AA starvation could provide an alternative strategy to augment the efficacy of vaccines against dengue and other infectious diseases.

Dissecting the heterogeneity of DENV vaccine-elicited cellular immunity using single-cell RNA sequencing and metabolic profiling.

Generating effective and durable T cell immunity is a critical prerequisite for vaccination against dengue virus (DENV) and other viral diseases. However, understanding the molecular mechanisms of vaccine-elicited T cell immunity remains a critical knowledge gap in vaccinology. In this study, we utilize single-cell RNA sequencing (scRNAseq) and longitudinal TCR clonotype analysis to identify a unique transcriptional signature present in acutely activated and clonally-expanded T cells that become committed to the memory repertoire. This effector/memory-associated transcriptional signature is dominated by a robust metabolic transcriptional program. Based on this transcriptional signature, we are able to define a set of markers that identify the most durable vaccine-reactive memory-precursor CD8+ T cells. This study illustrates the power of scRNAseq as an analytical tool to assess the molecular mechanisms of host control and vaccine modality in determining the magnitude, diversity and persistence of vaccine-elicited cell-mediated immunity.

Systematic review of dengue vaccine efficacy.

BACKGROUND: Dengue is an arbovirus that has rapidly spread worldwide, and the incidence of dengue has greatly increased in recent decades. The actual numbers of dengue cases are underreported, and many cases are not classified correctly. Recent estimates indicate that 390 million dengue infections occur per year (95% CI, 284-528 million), of which 96 million (67-136 million) are symptomatic infections of any severity. One of the goals of the World Health Organization is to reduce dengue mortality by 50% by the year 2020. The use of a vaccine can be an important strategy to achieve this goal. Vaccines for dengue are in various stages of development; in Brazil, only one commercial formulation is available (CYD-TDV), which was developed by Sanofi Pasteur. METHODS: To evaluate the efficacy of Dengue vaccine, a systematic review with a meta-analysis was conducted using randomized controlled clinical trials published between 2000 and 2017 that were identified in the MEDLINE databases via PubMed, LILACS, Cochrane Library, and EMBASE. The selection was performed by two reviewers independently, with disagreements resolved by a third reviewer. RESULTS: Seven clinical trials were included, with a total of 36,371 participants (66,511 person-years) between the ages of 2 and 45 years. The meta-analysis using the random-effects model estimated the efficacy of the vaccine at 44%, with a range from 25 to 59% and high heterogeneity (I2 = 80.1%). The serotype-stratified meta-analysis was homogeneous, except for serotype 2, with the heterogeneity of 64.5%. Most of the vaccinated individuals had previous immunity for at least one serotype, which generated safety concerns in individuals without previous immunity. CONCLUSIONS: Compared with other commercially available vaccines, the dengue vaccine showed poor efficacy.

The Conserved Molecular Determinants of Virulence in Dengue Virus.

Dengue virus belongs to the Flaviviridae family which also includes viruses such as the Zika, West Nile and yellow fever virus. Dengue virus generally causes mild disease, however, more severe forms of the dengue virus infection, dengue haemorrhagic fever (DHF) and dengue haemorrhagic fever with shock syndrome (DSS) can also occur, resulting in multiple organ failure and even death, especially in children. The only dengue vaccine available in the market, CYD-TDV offers limited coverage for vaccinees from 9-45 years of age and is only recommended for individuals with prior dengue exposure. A number of mutations that were shown to attenuate virulence of dengue virus in vitro and/or in vivo have been identified in the literature. The mutations which fall within the conserved regions of all four dengue serotypes are discussed. This review hopes to provide information leading to the construction of a live attenuated dengue vaccine that is suitable for all ages, irrespective of the infecting dengue serotype and prior dengue exposure.

Periods of high dengue transmission defined by rainfall do not impact efficacy of dengue vaccine in regions of endemic disease.

OBJECTIVE: To evaluate the association of rainy season with overall dengue disease incidence and with the efficacy of the Sanofi Pasteur recombinant, live, attenuated, tetravalent vaccine (CYD-TDV) in two randomized, controlled multicenter phase III clinical trials in Asia and Latin America. METHODS: Rainy seasons were defined for each study site using climatological information from the World Meteorological Organization. The dengue attack rate in the placebo group for each study month was calculated as the number of symptomatic, virologically-confirmed dengue events in a given month divided by the number of participants at risk in the same month. Time-dependent Cox proportional hazard models were used to test whether rainy season was associated with dengue disease and whether it modified vaccine efficacy in each of the two trials and in both of the trials combined. FINDINGS: Rainy season, country, and age were all significantly associated with dengue disease in both studies. Vaccine efficacy did not change during the rainy season in any of the analyses. CONCLUSIONS: Although dengue transmission and exposure are expected to increase during the rainy season, our results indicate that CYD-TDV vaccine efficacy remains constant throughout the year in endemic regions.

Dengue Fever: A Worldwide Threat An Overview of the Infection Process, Environmental Factors for a Global Outbreak, Diagnostic Platforms and Vaccine Developments.

Current review article focuses on dengue, which is one of the most fatal infectious illnesses and is considered to be a worldwide threat. The paper covers essential topics including an overview on neglected tropical diseases with specific emphasis on Dengue fever, mosquito's cycle of life and mechanism of infection, adaptive response, and different stages in dengue immunopathogenesis. The current work is also dedicated to the thorough study of dengue outbreak across the globe with a narrowed study to tropical and subtropical regions. Moreover, this review article demonstrates the correlation between climate factors and dengue incidence. Furthermore, we present an overview of the detection strategies of dengue including the latest developments in commercial and non-commercial platforms. Several attempts in developing an effective vaccine to protect individuals from dengue infection and the stage of clinical trials are gathered in the present work as well. Future directions including bio-control are also discussed in this review article. In an overall view, effective management of Dengue is a multidisciplinary task that requires international involvement from different backgrounds and expertise to address this global concern. This review article briefly portrays some of these connecting areas across the disciplines while many other perspectives remain uncovered.

Antibodies Against Modified NS1 Wing Domain Peptide Protect Against Dengue Virus Infection.

Dengue is the most common mosquito-transmitted viral infection for which an improved vaccine is still needed. Although nonstructural protein-1 (NS1) immunization can protect mice against dengue infection, molecular mimicry between NS1 and host proteins makes NS1-based vaccines challenging to develop. Based on the epitope recognized by the anti-NS1 monoclonal Ab (mAb) 33D2 which recognizes a conserved NS1 wing domain (NS1-WD) region but not host proteins, we synthesized a modified NS1-WD peptide to immunize mice. We found that both mAb 33D2 and modified NS1-WD peptide immune sera could induce complement-dependent lysis of dengue-infected but not un-infected cells in vitro. Furthermore, either active immunization with the modified NS1-WD peptide or passive transfer of mAb 33D2 efficiently protected mice against all serotypes of dengue virus infection. More importantly, dengue patients with more antibodies recognized the modified NS1-WD peptide had less severe disease. Thus, the modified NS1-WD peptide is a promising dengue vaccine candidate.

Molecular engineering and plant expression of an immunoglobulin heavy chain scaffold for delivery of a dengue vaccine candidate.

In order to enhance vaccine uptake by the immune cells in vivo, molecular engineering approach was employed to construct a polymeric immunoglobulin G scaffold (PIGS) that incorporates multiple copies of an antigen and targets the Fc gamma receptors on antigen-presenting cells. These self-adjuvanting immunogens were tested in the context of dengue infection, for which there is currently no globally licensed vaccine yet. Thus, the consensus domain III sequence (cEDIII) of dengue glycoprotein E was incorporated into PIGS and expressed in both tobacco plants and Chinese Ovary Hamster cells. Purified mouse and human cEDIII-PIGS were fractionated by HPLC into low and high molecular weight forms, corresponding to monomers, dimers and polymers. cEDIII-PIGS were shown to retain important Fc receptor functions associated with immunoglobulins, including binding to C1q component of the complement and the low affinity Fcγ receptor II, as well as to macrophage cells in vitro. These molecules were shown to be immunogenic in mice, with or without an adjuvant, inducing a high level IgG antibody response which showed a neutralizing potential against the dengue virus serotype 2. The cEDIII-PIGS also induced a significant cellular immune response, IFN-γ production and polyfunctional T cells in both the CD4+ and CD8+ compartments. This proof-of-principle study shows that the potent antibody Fc-mediated cellular functions can be harnessed to improve vaccine design, underscoring the potential of this technology to induce and modulate a broad-ranging immune response.

A Tetravalent Sub-unit Dengue Vaccine Formulated with Ionizable Cationic Lipid Nanoparticle induces Significant Immune Responses in Rodents and Non-Human Primates.

Dengue virus has emerged as an important arboviral infection worldwide. As a complex pathogen, with four distinct serotypes, the development of a successful Dengue virus vaccine has proven to be challenging. Here, we describe a novel Dengue vaccine candidate that contains truncated, recombinant, Dengue virus envelope protein from all four Dengue virus serotypes (DEN-80E) formulated with ionizable cationic lipid nanoparticles (LNPs). Immunization studies in mice, Guinea pigs, and in Rhesus macaques, revealed that LNPs induced high titers of Dengue virus neutralizing antibodies, with or without co-administration or encapsulation of a Toll-Like Receptor 9 agonist. Importantly, LNPs were also able to boost DEN-80E specific CD4+ and CD8+ T cell responses. Cytokine and chemokine profiling revealed that LNPs induced strong chemokine responses without significant induction of inflammatory cytokines. In addition to being highly efficacious, the vaccine formulation proved to be well-tolerated, demonstrating no elevation in any of the safety parameters evaluated. Notably, reduction in cationic lipid content of the nanoparticle dramatically reduced the LNP's ability to boost DEN-80E specific immune responses, highlighting the crucial role for the charge of the LNP. Overall, our novel studies, across multiple species, reveal a promising tetravalent Dengue virus sub-unit vaccine candidate.

The Potential Impact of Vaccination on the Dynamics of Dengue Infections.

Dengue, classified as a 'neglected topical disease', is currently regarded globally as the most important mosquito-borne viral disease, which inflicts substantial socioeconomic and health burden in many tropical and subtropical regions of the world. While efforts continue towards developing and improving the efficacy of a tetravalent vaccine to protect individuals against all dengue virus serotypes, the long-term epidemiological impact of vaccination remains elusive. We develop a serotype-specific, vector-host compartmental model to evaluate the effect of vaccination in the presence of antibody-dependent enhancement and cross-protection following recovery from primary infection. Reproducing the reported multi-annual patterns of dengue infection, our model projects that vaccination can dramatically reduce the overall incidence of the disease. However, if the duration of vaccine-induced protection is shorter than the average lifetime of the human population, vaccination can potentially increase the incidence of severe infection of dengue haemorrhagic fever due to the effects of antibody-dependent enhancement. The magnitude and timelines for this increase depend strongly on the efficacy and duration of the vaccine-induced protection. Corresponding to the current estimates of vaccine efficacy, we show that dengue eradication is infeasible using an imperfect vaccine. Furthermore, for a vaccine that induces lifetime protection, a nearly full coverage of infant vaccination is required for dengue elimination. Our findings suggest that other vector control measures may still play a significant role in dengue prevention even when a vaccine with high protection efficacy becomes available.

Role of humoral versus cellular responses induced by a protective dengue vaccine candidate.

With 2.5 billion people at risk, dengue is a major emerging disease threat and an escalating public health problem worldwide. Dengue virus causes disease ranging from a self-limiting febrile illness (dengue fever) to the potentially fatal dengue hemorrhagic fever/dengue shock syndrome. Severe dengue disease is associated with sub-protective levels of antibody, which exacerbate disease upon re-infection. A dengue vaccine should generate protective immunity without increasing severity of disease. To date, the determinants of vaccine-mediated protection against dengue remain unclear, and additional correlates of protection are urgently needed. Here, mice were immunized with viral replicon particles expressing the dengue envelope protein ectodomain to assess the relative contribution of humoral versus cellular immunity to protection. Vaccination with viral replicon particles provided robust protection against dengue challenge. Vaccine-induced humoral responses had the potential to either protect from or exacerbate dengue disease upon challenge, whereas cellular immune responses were beneficial. This study explores the immunological basis of protection induced by a dengue vaccine and suggests that a safe and efficient vaccine against dengue should trigger both arms of the immune system.

Acceptability of hypothetical dengue vaccines among travelers.

BACKGROUND: Dengue viruses have spread widely in recent decades and cause tens of millions of infections mostly in tropical and subtropical areas. Vaccine candidates are being studied aggressively and may be ready for licensure soon. METHODS: We surveyed patients with past or upcoming travel to dengue-endemic countries to assess rates and determinants of acceptance for four hypothetical dengue vaccines with variable efficacy and adverse event (AE) profiles. Acceptance ratios were calculated for vaccines with varied efficacy and AE risk. RESULTS: Acceptance of the four hypothetical vaccines ranged from 54% for the vaccine with lower efficacy and serious AE risk to 95% for the vaccine with higher efficacy and minor AE risk. Given equal efficacy, vaccines with lower AE risk were better accepted than those with higher AE risk; given equivalent AE risk, vaccines with higher efficacy were better accepted than those with lower efficacy. History of Japanese encephalitis vaccination was associated with lower vaccine acceptance for one of the hypothetical vaccines. US-born travelers were more likely than non-US born travelers to accept a vaccine with 75% efficacy and a risk of minor AEs (p = 0.003). Compared with North American-born travelers, Asian- and African-born travelers were less likely to accept both vaccines with 75% efficacy. CONCLUSIONS: Most travelers would accept a safe and efficacious dengue vaccine if one were available. Travelers valued fewer potential AEs over increased vaccine efficacy.

Rational design of a live attenuated dengue vaccine: 2'-o-methyltransferase mutants are highly attenuated and immunogenic in mice and macaques.

Dengue virus is transmitted by Aedes mosquitoes and infects at least 100 million people every year. Progressive urbanization in Asia and South-Central America and the geographic expansion of Aedes mosquito habitats have accelerated the global spread of dengue, resulting in a continuously increasing number of cases. A cost-effective, safe vaccine conferring protection with ideally a single injection could stop dengue transmission. Current vaccine candidates require several booster injections or do not provide protection against all four serotypes. Here we demonstrate that dengue virus mutants lacking 2'-O-methyltransferase activity are highly sensitive to type I IFN inhibition. The mutant viruses are attenuated in mice and rhesus monkeys and elicit a strong adaptive immune response. Monkeys immunized with a single dose of 2'-O-methyltransferase mutant virus showed 100% sero-conversion even when a dose as low as 1,000 plaque forming units was administrated. Animals were fully protected against a homologous challenge. Furthermore, mosquitoes feeding on blood containing the mutant virus were not infected, whereas those feeding on blood containing wild-type virus were infected and thus able to transmit it. These results show the potential of 2'-O-methyltransferase mutant virus as a safe, rationally designed dengue vaccine that restrains itself due to the increased susceptibility to the host's innate immune response.

Diagnosis of dengue: an update.

Early diagnosis of dengue, the most common mosquito-borne disease globally, remains challenging. Dengue presents initially as undifferentiated fever, with symptoms becoming more pathognomonic in the later stages of illness. This limits the timeliness in the delivery of appropriate supportive interventions. Laboratory tests are useful for diagnosis although the short-lived viremia and the presence of secondary infection with one of the four heterologous viral serotypes collectively complicate the choice and interpretation of laboratory tests. In this article, the authors review the various approaches for diagnosis of dengue and discuss the appropriate tests to use, including when a dengue vaccine, which is in the late stages of development, is licensed for use. The ensuing reduced dengue prevalence could make diagnosis for vaccine efficacy and escape-mutant monitoring even more challenging.

Backward bifurcations in dengue transmission dynamics.

A deterministic model for the transmission dynamics of a strain of dengue disease, which allows transmission by exposed humans and mosquitoes, is developed and rigorously analysed. The model, consisting of seven mutually-exclusive compartments representing the human and vector dynamics, has a locally-asymptotically stable disease-free equilibrium (DFE) whenever a certain epidemiological threshold, known as the basic reproduction number(R(0)) is less than unity. Further, the model exhibits the phenomenon of backward bifurcation, where the stable DFE coexists with a stable endemic equilibrium. The epidemiological consequence of this phenomenon is that the classical epidemiological requirement of making R(0) less than unity is no longer sufficient, although necessary, for effectively controlling the spread of dengue in a community. The model is extended to incorporate an imperfect vaccine against the strain of dengue. Using the theory of centre manifold, the extended model is also shown to undergo backward bifurcation. In both the original and the extended models, it is shown, using Lyapunov function theory and LaSalle Invariance Principle, that the backward bifurcation phenomenon can be removed by substituting the associated standard incidence function with a mass action incidence. In other words, in addition to establishing the presence of backward bifurcation in models of dengue transmission, this study shows that the use of standard incidence in modelling dengue disease causes the backward bifurcation phenomenon of dengue disease.

Biological characteristics of dengue virus and potential targets for drug design.

Dengue infection is a major cause of morbidity in tropical and subtropical regions, bringing nearly 40% of the world population at risk and causing more than 20,000 deaths per year. But there is neither a vaccine for dengue disease nor antiviral drugs to treat the infection. In recent years, dengue infection has been particularly prevalent in India, Southeast Asia, Brazil, and Guangdong Province, China. In this article, we present a brief summary of the biological characteristics of dengue virus and associated flaviviruses, and outline the progress on studies of vaccines and drugs based on potential targets of the dengue virus.