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.

Efficacy of Wolbachia-Infected Mosquito Deployments for the Control of Dengue.

BACKGROUND: Aedes aegypti mosquitoes infected with the wMel strain of Wolbachia pipientis are less susceptible than wild-type A. aegypti to dengue virus infection. METHODS: We conducted a cluster-randomized trial involving releases of wMel-infected A. aegypti mosquitoes for the control of dengue in Yogyakarta, Indonesia. We randomly assigned 12 geographic clusters to receive deployments of wMel-infected A. aegypti (intervention clusters) and 12 clusters to receive no deployments (control clusters). All clusters practiced local mosquito-control measures as usual. A test-negative design was used to assess the efficacy of the intervention. Patients with acute undifferentiated fever who presented to local primary care clinics and were 3 to 45 years of age were recruited. Laboratory testing was used to identify participants who had virologically confirmed dengue (VCD) and those who were test-negative controls. The primary end point was symptomatic VCD of any severity caused by any dengue virus serotype. RESULTS: After successful introgression of wMel into the intervention clusters, 8144 participants were enrolled; 3721 lived in intervention clusters, and 4423 lived in control clusters. In the intention-to-treat analysis, VCD occurred in 67 of 2905 participants (2.3%) in the intervention clusters and in 318 of 3401 (9.4%) in the control clusters (aggregate odds ratio for VCD, 0.23; 95% confidence interval [CI], 0.15 to 0.35; P = 0.004). The protective efficacy of the intervention was 77.1% (95% CI, 65.3 to 84.9) and was similar against the four dengue virus serotypes. The incidence of hospitalization for VCD was lower among participants who lived in intervention clusters (13 of 2905 participants [0.4%]) than among those who lived in control clusters (102 of 3401 [3.0%]) (protective efficacy, 86.2%; 95% CI, 66.2 to 94.3). CONCLUSIONS: Introgression of wMel into A. aegypti populations was effective in reducing the incidence of symptomatic dengue and resulted in fewer hospitalizations for dengue among the participants. (Funded by the Tahija Foundation and others; AWED ClinicalTrials.gov number, NCT03055585; Indonesia Registry number, INA-A7OB6TW.).

Burden of Postinfectious Symptoms after Acute Dengue, Vietnam.

We assessed predominantly pediatric patients in Vietnam with dengue and other febrile illness 3 months after acute illness. Among dengue patients, 47% reported >1 postacute symptom. Most resolved by 3 months, but alopecia and vision problems often persisted. Our findings provide additional evidence on postacute dengue burden and confirm children are affected.

Multicore fiber beacon system for reducing back-reflection in satellite quantum key distribution.

Optical beaconing is an important part of the acquisition, pointing and tracking system necessary for free-space quantum key distribution (QKD). However, uplink beacon back-reflections from the receiver architecture can result in noise. Wavelength- and time-division multiplexing has been used, but neither is yet sufficient to make back-reflection negligible. The use of additional telescopes increases complexity and pointing error. Here, we propose the use of a 2-by-2 multicore fiber, to act as an optical uplink beacon source. This spatially separates the QKD channel and optical uplink beacon. Up to 50 dB improvement in noise rejection over a purely spectrally divided system was demonstrated. The route to further improvements through greater fiber core separation is described. Beacon systems designed in this way could provide a combination of reduced complexity and improved noise performance to free-space and satellite QKD and optical communications.

Modulation of acyl-carnitines, the broad mechanism behind Wolbachia-mediated inhibition of medically important flaviviruses in Aedes aegypti.

Wolbachia-infected mosquitoes are refractory to flavivirus infections, but the role of lipids in Wolbachia-mediated virus blocking remains to be elucidated. Here, we use liquid chromatography mass spectrometry to provide a comprehensive picture of the lipidome of Aedes aegypti (Aag2) cells infected with Wolbachia only, either dengue or Zika virus only, and Wolbachia-infected Aag2 cells superinfected with either dengue or Zika virus. This approach identifies a class of lipids, acyl-carnitines, as being down-regulated during Wolbachia infection. Furthermore, treatment with an acyl-carnitine inhibitor assigns a crucial role for acyl-carnitines in the replication of dengue and Zika viruses. In contrast, depletion of acyl-carnitines increases Wolbachia density while addition of commercially available acyl-carnitines impairs Wolbachia production. Finally, we show an increase in flavivirus infection of Wolbachia-infected cells with the addition of acyl-carnitines. This study uncovers a previously unknown role for acyl-carnitines in this tripartite interaction that suggests an important and broad mechanism that underpins Wolbachia-mediated pathogen blocking.

Using Wolbachia to Eliminate Dengue: Will the Virus Fight Back?

Recent field trials have demonstrated that dengue incidence can be substantially reduced by introgressing strains of the endosymbiotic bacterium Wolbachia into Aedes aegypti mosquito populations. This strategy relies on Wolbachia reducing the susceptibility of Ae. aegypti to disseminated infection by positive-sense RNA viruses like dengue. However, RNA viruses are well known to adapt to antiviral pressures. Here, we review the viral infection stages where selection for Wolbachia-resistant virus variants could occur. We also consider the genetic constraints imposed on viruses that alternate between vertebrate and invertebrate hosts, and the likely selection pressures to which dengue virus might adapt in order to be effectively transmitted by Ae. aegypti that carry Wolbachia. While there are hurdles to dengue viruses developing resistance to Wolbachia, we suggest that long-term surveillance for resistant viruses should be an integral component of Wolbachia-introgression biocontrol programs.

Novel phenotype of Wolbachia strain wPip in Aedes aegypti challenges assumptions on mechanisms of Wolbachia-mediated dengue virus inhibition.

The bacterial endosymbiont Wolbachia is a biocontrol tool that inhibits the ability of the Aedes aegypti mosquito to transmit positive-sense RNA viruses such as dengue and Zika. Growing evidence indicates that when Wolbachia strains wMel or wAlbB are introduced into local mosquito populations, human dengue incidence is reduced. Despite the success of this novel intervention, we still do not fully understand how Wolbachia protects mosquitoes from viral infection. Here, we demonstrate that the Wolbachia strain wPip does not inhibit virus infection in Ae. aegypti. We have leveraged this novel finding, and a panel of Ae. aegypti lines carrying virus-inhibitory (wMel and wAlbB) and non-inhibitory (wPip) strains in a common genetic background, to rigorously test a number of hypotheses about the mechanism of Wolbachia-mediated virus inhibition. We demonstrate that, contrary to previous suggestions, there is no association between a strain's ability to inhibit dengue infection in the mosquito and either its typical density in the midgut or salivary glands, or the degree to which it elevates innate immune response pathways in the mosquito. These findings, and the experimental platform provided by this panel of genetically comparable mosquito lines, clear the way for future investigations to define how Wolbachia prevents Ae. aegypti from transmitting viruses.

Multiple Wolbachia strains provide comparative levels of protection against dengue virus infection in Aedes aegypti.

The insect bacterium Wolbachia pipientis is being introgressed into Aedes aegypti populations as an intervention against the transmission of medically important arboviruses. Here we compare Ae. aegypti mosquitoes infected with wMelCS or wAlbB to the widely used wMel Wolbachia strain on an Australian nuclear genetic background for their susceptibility to infection by dengue virus (DENV) genotypes spanning all four serotypes. All Wolbachia-infected mosquitoes were more resistant to intrathoracic DENV challenge than their wildtype counterparts. Blocking of DENV replication was greatest by wMelCS. Conversely, wAlbB-infected mosquitoes were more susceptible to whole body infection than wMel and wMelCS. We extended these findings via mosquito oral feeding experiments, using viremic blood from 36 acute, hospitalised dengue cases in Vietnam, additionally including wMel and wildtype mosquitoes on a Vietnamese nuclear genetic background. As above, wAlbB was less effective at blocking DENV replication in the abdomen compared to wMel and wMelCS. The transmission potential of all Wolbachia-infected mosquito lines (measured by the presence/absence of infectious DENV in mosquito saliva) after 14 days, was significantly reduced compared to their wildtype counterparts, and lowest for wMelCS and wAlbB. These data support the use of wAlbB and wMelCS strains for introgression field trials and the biocontrol of DENV transmission. Furthermore, despite observing significant differences in transmission potential between wildtype mosquitoes from Australia and Vietnam, no difference was observed between wMel-infected mosquitoes from each background suggesting that Wolbachia may override any underlying variation in DENV transmission potential.

Higher Plasma Viremia in the Febrile Phase Is Associated With Adverse Dengue Outcomes Irrespective of Infecting Serotype or Host Immune Status: An Analysis of 5642 Vietnamese Cases.

BACKGROUND: One of the generally accepted constructs of dengue pathogenesis is that clinical disease severity is at least partially dependent upon plasma viremia, yet data on plasma viremia in primary versus secondary infections and in relation to clinically relevant endpoints remain limited and contradictory. METHODS: Using a large database comprising detailed clinical and laboratory characterization of Vietnamese participants enrolled in a series of research studies executed over a 15-year period, we explored relationships between plasma viremia measured by reverse transcription-polymerase chain reaction and 3 clinically relevant endpoints-severe dengue, plasma leakage, and hospitalization-in the dengue-confirmed cases. All 4 dengue serotypes and both primary and secondary infections were well represented. In our logistic regression models we allowed for a nonlinear effect of viremia and for associations between viremia and outcome to differ by age, serotype, host immune status, and illness day at study enrollment. RESULTS: Among 5642 dengue-confirmed cases we identified 259 (4.6%) severe dengue cases, 701 (12.4%) patients with plasma leakage, and 1441 of 4008 (40.0%) patients recruited in outpatient settings who were subsequently hospitalized. From the early febrile phase onwards, higher viremia increased the risk of developing all 3 endpoints, but effect sizes were modest (ORs ranging from 1.12-1.27 per 1-log increase) compared with the effects of a secondary immune response (ORs, 1.67-7.76). The associations were consistent across age, serotype, and immune status groups, and in the various sensitivity and subgroup analyses we undertook. CONCLUSIONS: Higher plasma viremia is associated with increased dengue severity, regardless of serotype or immune status.

Flavivirus replication kinetics in early-term placental cell lines with different differentiation pathways.

BACKGROUND: The uncontrollable spread of Zika virus (ZIKV) in the Americas during 2015-2017, and its causal link to microcephaly in newborns and Guillain-Barré syndrome in adults, led the World Health Organisation to declare it a global public health emergency. One of the most notable features of ZIKV pathogenesis was the ability of the virus to pass the placental barrier to infect the growing foetus. This pathogenic trait had not been observed previously for medically important flaviviruses, including dengue and yellow fever viruses. METHODS: In this study we evaluated the replication kinetics of ZIKV and the related encephalitic flavivirus West Nile strain Kunjin virus (WNVKUN) in early-term placental cell lines. RESULTS: We have observed that WNVKUN in fact replicates with a greater rate and to higher titres that ZIKV in these cell lines. CONCLUSIONS: These results would indicate the potential for all flaviviruses to replicate in placental tissue but it is the ability to cross the placenta itself that is the restrictive factor in the clinical progression and presentation of congenital Zika syndrome.

Viral genetic diversity and protective efficacy of a tetravalent dengue vaccine in two phase 3 trials.

Two phase 3 placebo-controlled trials of the CYD-TDV vaccine, evaluated in children aged 2-14 y (CYD14) and 9-16 y (CYD15), demonstrated vaccine efficacy (VE) of 56.5% and 60.8%, respectively, against symptomatic virologically confirmed dengue (VCD). Sieve analyses were conducted to evaluate whether and how VE varied with amino acid sequence features of dengue viruses (DENVs). DENV premembrane/envelope amino acid sequences from VCD endpoint cases were aligned with the vaccine insert sequences, and extensions of the proportional hazards model were applied to assess variation in VE with amino acid mismatch proportion distances from vaccine strains, individual amino acid residues, and phylogenetic genotypes. In CYD14, VE against VCD of any serotype (DENV-Any) decreased significantly with increasing amino acid distance from the vaccine, whereas in CYD15, VE against DENV-Any was distance-invariant. Restricting to the common age range and amino acid distance range between the trials and accounting for differential VE by serotype, however, showed no evidence of VE variation with distance in either trial. In serotype-specific analyses, VE against DENV4 decreased significantly with increasing amino acid distance from the DENV4 vaccine insert and was significantly greater against residue-matched DENV4 at eight signature positions. These effects were restricted to 2- to 8-y-olds, potentially because greater seropositivity of older children at baseline might facilitate a broader protective immune response. The relevance of an antigenic match between vaccine strains and circulating DENVs was also supported by greater estimated VE against serotypes and genotypes for which the circulating DENVs had shorter amino acid sequence distances from the vaccine.

Field- and clinically derived estimates of Wolbachia-mediated blocking of dengue virus transmission potential in Aedes aegypti mosquitoes.

The wMel strain of Wolbachia can reduce the permissiveness of Aedes aegypti mosquitoes to disseminated arboviral infections. Here, we report that wMel-infected Ae. aegypti (Ho Chi Minh City background), when directly blood-fed on 141 viremic dengue patients, have lower dengue virus (DENV) transmission potential and have a longer extrinsic incubation period than their wild-type counterparts. The wMel-infected mosquitoes that are field-reared have even greater relative resistance to DENV infection when fed on patient-derived viremic blood meals. This is explained by an increased susceptibility of field-reared wild-type mosquitoes to infection than laboratory-reared counterparts. Collectively, these field- and clinically relevant findings support the continued careful field-testing of wMel introgression for the biocontrol of Ae. aegypti-born arboviruses.

Analysis of cluster-randomized test-negative designs: cluster-level methods.

Intervention trials of vector control methods often require community level randomization with appropriate inferential methods. For many interventions, the possibility of confounding due to the effects of health-care seeking behavior on disease ascertainment remains a concern. The test-negative design, a variant of the case-control method, was introduced to mitigate this issue in the assessment of the efficacy of influenza vaccination (measured at an individual level) on influenza infection. Here, we introduce a cluster-randomized test-negative design that includes randomization of the intervention at a group level. We propose several methods for estimation and inference regarding the relative risk (RR). The inferential methods considered are based on the randomization distribution induced by permuting intervention assignment across two sets of randomly selected clusters. The motivating example is a current study of the efficacy of randomized releases of Wolbachia-infected Aedes aegypti mosquitoes to reduce the incidence of dengue in Yogyakarta City, Indonesia. Estimation and inference techniques are assessed through a simulation study.

C-reactive protein as a potential biomarker for disease progression in dengue: a multi-country observational study.

BACKGROUND: Dengue infection can cause a wide spectrum of clinical outcomes. The severe clinical manifestations occur sufficiently late in the disease course, during day 4-6 of illness, to allow a window of opportunity for risk stratification. Markers of inflammation may be useful biomarkers. We investigated the value of C-reactive protein (CRP) measured early on illness days 1-3 to predict dengue disease outcome and the difference in CRP levels between dengue and other febrile illnesses (OFI). METHOD: We performed a nested case-control study using the clinical data and samples collected from the IDAMS-consortium multi-country study. This was a prospective multi-center observational study that enrolled almost 8000 participants presenting with a dengue-like illness to outpatient facilities in 8 countries across Asia and Latin America. Predefined severity definitions of severe and intermediate dengue were used as the primary outcomes. A total of 281 cases with severe/intermediate dengue were compared to 836 uncomplicated dengue patients as controls (ratio 1:3), and also 394 patients with OFI. RESULTS: In patients with confirmed dengue, median (interquartile range) of CRP level within the first 3 days was 30.2 mg/L (12.4-61.2 mg/L) (uncomplicated dengue, 28.6 (10.5-58.9); severe or intermediate dengue, 34.0 (17.4-71.8)). Higher CRP levels in the first 3 days of illness were associated with a higher risk of severe or intermediate outcome (OR 1.17, 95% CI 1.07-1.29), especially in children. Higher CRP levels, exceeding 30 mg/L, also associated with hospitalization (OR 1.37, 95% CI 1.14-1.64) and longer fever clearance time (HR 0.84, 95% CI 0.76-0.93), especially in adults. CRP levels in patients with dengue were higher than patients with potential viral infection but lower than patients with potential bacterial infection, resulting in a quadratic association between dengue diagnosis and CRP, with levels of approximately 30 mg/L associated with the highest risk of having dengue. CRP had a positive correlation with total white cell count and neutrophils and negative correlation with lymphocytes, but did not correlate with liver transaminases, albumin, or platelet nadir. CONCLUSIONS: In summary, CRP measured in the first 3 days of illness could be a useful biomarker for early dengue risk prediction and may assist differentiating dengue from other febrile illnesses.

The Role of Maternally Acquired Antibody in Providing Protective Immunity Against Nontyphoidal Salmonella in Urban Vietnamese Infants: A Birth Cohort Study.

Background: Nontyphoidal Salmonella (NTS) organisms are a major cause of gastroenteritis and bacteremia, but little is known about maternally acquired immunity and natural exposure in infant populations residing in areas where NTS disease is highly endemic. Methods: We recruited 503 pregnant mothers and their infants (following delivery) from urban areas in Vietnam and followed infants until they were 1 year old. Exposure to the dominant NTS serovars, Salmonella enterica serovars Typhimurium and Enteritidis, were assessed using lipopolysaccharide (LPS) O antigen-specific antibodies. Antibody dynamics, the role of maternally acquired antibodies, and NTS seroincidence rates were modeled using multivariate linear risk factor models and generalized additive mixed-effect models. Results: Transplacental transfer of NTS LPS-specific maternal antibodies to infants was highly efficient. Waning of transplacentally acquired NTS LPS-specific antibodies at 4 months of age left infants susceptible to Salmonella organisms, after which they began to seroconvert. High seroincidences of S. Typhimurium and S. Enteritidis LPS were observed, and infants born with higher anti-LPS titers had greater plasma bactericidal activity and longer protection from seroconversion. Conclusions: Although Vietnamese infants have extensive exposure to NTS, maternally acquired antibodies appear to play a protective role against NTS infections during early infancy. These findings suggest that prenatal immunization may be an appropriate strategy to protect vulnerable infants from NTS disease.

The global distribution and burden of dengue.

Dengue is a systemic viral infection transmitted between humans by Aedes mosquitoes. For some patients, dengue is a life-threatening illness. There are currently no licensed vaccines or specific therapeutics, and substantial vector control efforts have not stopped its rapid emergence and global spread. The contemporary worldwide distribution of the risk of dengue virus infection and its public health burden are poorly known. Here we undertake an exhaustive assembly of known records of dengue occurrence worldwide, and use a formal modelling framework to map the global distribution of dengue risk. We then pair the resulting risk map with detailed longitudinal information from dengue cohort studies and population surfaces to infer the public health burden of dengue in 2010. We predict dengue to be ubiquitous throughout the tropics, with local spatial variations in risk influenced strongly by rainfall, temperature and the degree of urbanization. Using cartographic approaches, we estimate there to be 390 million (95% credible interval 284-528) dengue infections per year, of which 96 million (67-136) manifest apparently (any level of disease severity). This infection total is more than three times the dengue burden estimate of the World Health Organization. Stratification of our estimates by country allows comparison with national dengue reporting, after taking into account the probability of an apparent infection being formally reported. The most notable differences are discussed. These new risk maps and infection estimates provide novel insights into the global, regional and national public health burden imposed by dengue. We anticipate that they will provide a starting point for a wider discussion about the global impact of this disease and will help to guide improvements in disease control strategies using vaccine, drug and vector control methods, and in their economic evaluation.

Dengue-Associated Posterior Reversible Encephalopathy Syndrome, Vietnam.

Dengue can cause neurologic complications in addition to the more common manifestations of plasma leakage and coagulopathy. Posterior reversible encephalopathy syndrome has rarely been described in dengue, although the pathophysiology of endothelial dysfunction likely underlies both. We describe a case of dengue-associated posterior reversible encephalopathy syndrome and discuss diagnosis and management.

Cluster-Randomized Test-Negative Design Trials: A Novel and Efficient Method to Assess the Efficacy of Community-Level Dengue Interventions.

Cluster-randomized controlled trials are the gold standard for assessing efficacy of community-level interventions, such as vector-control strategies against dengue. We describe a novel cluster-randomized trial methodology with a test-negative design (CR-TND), which offers advantages over traditional approaches. This method uses outcome-based sampling of patients presenting with a syndrome consistent with the disease of interest, who are subsequently classified as test-positive cases or test-negative controls on the basis of diagnostic testing. We used simulations of a cluster trial to demonstrate validity of efficacy estimates under the test-negative approach. We demonstrated that, provided study arms are balanced for both test-negative and test-positive illness at baseline and that other test-negative design assumptions are met, the efficacy estimates closely match true efficacy. Analytical considerations for an odds ratio-based effect estimate arising from clustered data and potential approaches to analysis are also discussed briefly. We concluded that application of the test-negative design to certain cluster-randomized trials could increase their efficiency and ease of implementation.