Community Research and Development Information Service - CORDIS


DENFREE Report Summary

Project ID: 282378
Funded under: FP7-HEALTH
Country: France

Periodic Report Summary 3 - DENFREE (Dengue research Framework for Resisting Epidemics in Europe)

Project Context and Objectives:
Project context
Dengue is a major international public health concern and one of the most important arthropod borne diseases. Approximately 55% of world’s population is at risk of dengue virus (DENV) infection.
The current state-of-the-art strategy to manage the burden of dengue in countries with endemic DENV has focused on improving clinical care of hospitalized severe cases. Whilst this has reduced the case fatality rate, alone it is insufficient in the face of an ever-increasing population at risk. Mosquito control, although successful in some cases, has largely failed to make an impact over the long term. Epidemiological studies have shown that most DENV infections are asymptomatic or subclinical and thus in a completely naïve population, the first hospital cases will be the tip of the iceberg. Hospital-based surveillance will be inadequate – too little and too late.
Currently we know very little about the epidemiology of subclinical infections. How frequent are they? What factors determine the symptomatic/asymptomatic outcome of infection? Can they infect mosquitoes? Is the duration of infection different from symptomatic episodes? The role of individuals with asymptomatic infection in spreading the virus must be addressed. Such epidemiological information is crucial to predict local dengue epidemiology and determine whether intervention strategies, such as anti-viral treatment targeting asymptomatics, are useful.
DENFREE combines expertise from different disciplines. Each participant generates expert data using samples from the same individuals with well-characterized clinical and epidemiological data. This multidisciplinary and complementary approach will allow each participant to access to data in other relevant fields and analyze them together. From the knowledge acquired, we can make serious efforts at reducing the impact of dengue in endemic areas and at preventing its extension into new areas.
Project objectives
The overall objective of DENFREE program is to understand a role of asymptomatic dengue infection in dengue transmission and their virological and immunological profile. We plan to estimate risk of having dengue outbreaks in Europe. We documented climate prediction models, European vector competence, viral genetic markers for adaptation to European mosquitoes and population genetic susceptibility to clinical and severe dengue. In addition, we developed new tools for point of care diagnostic test for detecting asymptomatic DENV infected individuals, tested new potential therapeutic agents, identified new vaccine candidates and evaluated innovative vector control strategies suitable for European countries. The majority of the tasks have been finished and the aims achieved.
In this 3rd reporting period, the aims were:
1. To finish predictive models under invasive scenarios and intervention
2. To finish agent based models describing dengue epidemic diffusion
3. To finish identification of viral genetic markers determining susceptibility to European vectors
4. To evaluate new diagnostic tools using samples collected from patients
5. To finish functional and structural characterization of human monoclonal antibodies
6. To finish immunological characterization of asymptomatic DENV infection vs clinical dengue.
5. To finish human genetic study to identify genetic markers, genes and pathways involved in susceptibility to clinical and severe dengue.

Project Results:
Both field study sites successfully accomplished their missions of generating biological samples within defined epidemiological conditions for use by the other studies.
Diagnostic test:
We have published an isothermal amplification method – reverse transcription- Recombinase Polymerase Amplification to detect DENV genome in clinical specimens. During this period, we have developed DNA-tagged Dengue antigen for antibody detection method. The road-block of DNA-antigen cross-linking has been overcome and antigens have been DNA-coded. Amplified products are now detected sensitively by fluorescence. This will allow a highly sensitive test for DENV antibodies and will be combined with a virus type specific amplification method in the future.
Calibrated climate dataset:
WP3 has generated a calibrated climate dataset that can extensively be used by other WPs and projects, performed database inter-comparison for the different climate product variables and created a user-friendly tutorial (see Climate Explorer tutorial posted in the DENFREE website). The tutorial intends to illustrate how even a naive user can visualize and download different types of climate observations and simulations for research purposes. Similarly, an online near-operational skill finder tool has been developed that can be used to assess where and in which seasons climate information is going to be most useful for disease prediction purposes.
Descriptive and Predictive models:
In WP4 we developed models and improved and extended already existing models including include secondary infection, temporary cross-immunity, seasonality, climatic variables and vector dynamics. We then explored the effect of vaccine intervention through our dynamical modelling approach. Because of the non-protective effect of the vaccine in naïve individuals, we recommend a screening on sero-status before administering the vaccine. Using optimal control methods we showed that such methods can now be used to evaluate optimal controls via vaccination and mosquito control where both methods alone have their limitations.
The spatial-temporal pattern of propagation of annual epidemics in Cambodia highlighted the role that human movement plays. Particularly we revealed a great spatial heterogeneity in the propagation of the annual epidemic. Each year, epidemics are highly synchronous over a large geographic area along the busiest national road of the country whereas travelling waves emanate from few rural areas and move slowly along the Mekong River towards the capital, Phnom Penh. Then, we have implemented a simplified ABM/metapopulation mixed model where a single strain vector borne infection travelling through a network of cities and villages is deployed. A major finding is that a complete stationary arrest of symptomatics has little impact on the peak of disease incidence at virtually every ratio of symptomatic-asymptomatic infected individuals in the population.
At a larger geographical scale, we generated a database of dengue cases in French Polynesia and demonstrated that maintenance of dengue can only occur by island hopping; even the large island of Tahiti is unable to maintain the virus for extended periods and thus the virus persists in this island complex by hopping from one island to the next.
Finally we established a database with climate information for improving Public Health decision making and validated this with dengue data from southern coastal Ecuador. Oceanic Niño Index (ONI), rainfall and temperature were positively associated with dengue and the ONI was identified as the most important climate variable. Our structural time series model is specifically tailored to forecast at long lead times of 24 months or more, going well beyond the traditional ¨spring barrier¨ of ENSO prediction, thus generating more lead time for prediction and preparedness.
Asymptomatic transmission, European vector competence and innovative vector control strategy
Most of dengue human cases are asymptomatic or result in too few symptoms to be detected by existing surveillance systems. These clinically inapparent cases were not considered to develop a high enough viremia to infect mosquitoes and therefore were assumed to be dead-ends for virus transmission. We demonstrated that not only asymptomatic people are infectious to mosquitoes, but also that they are significantly more infectious to mosquitoes than people with symptomatic infections at a given viremia level (Duong et al. 2015 PNAS).
In Europe, the Asian tiger mosquito has been introduced in 1990 and is now present in 20 European countries and was responsible for local dengue cases in France, Croatia, and Madeira. Owing to the increasing number of autochthonous dengue cases, we evaluated the ability of European populations of Ae. albopictus to transmit imported strains of DENV. We showed that (i) viral dissemination increased with the dpi, (ii) viral dissemination was similar whatever the DENV-1 (France or Thailand), and (iii) differences were associated with the mosquito population.
Aedes albopictus is not considered as a main vector of DENV. However owing to the recurrent detections of local dengue cases in Europe, the question of its long-term adaption for a better transmission of DENV has been addressed. We have designed a protocol to experimentally enhance transmission potential of DENV for European Ae. albopictus. When the viruses were passaged in Ae. albopictus mosquitoes and cell culture alternatively, some new variants reached consensus level (frequency > 50%). The variant reached consensus level within two passages following initial detection and became almost fixed (frequency > 99%) soon after, consistent with a strong selective sweep.
The auto-dissemination of the insect growth hormone, pyriproxyfen (PPF) was tested in Madeira Island, through a small pilot study in the village of Paul do Mar and Funchal. PPF implementation led to ~20% higher larval mortality rates in sentinel sites but an even greater reduction in adult densities.
Viral genetic diversity and adaptation
Concerning viral population study using DENV-3 samples collected at different time points during the 2001-2002 Cuban epidemic, it was demonstrated that greater variability occurred in the non-structural genes compared with structural genes, in terms of significant minority variants, and particularly towards the end of the epidemic. Still greater variability was observed in the non-structural genes compared with structural genes; but it was noteworthy that patients with secondary infection showed greater variability than patients with primary infections. In addition, secondary patients presented minority variants in the structural genes (PrM and E), some of which were non-synonymous. By contrast, patients suffering primary infections only had mostly synonymous minority variants (>1%) in non-structural genes. Our results suggest that changes in the viral population swarm occurred with the epidemic’s progression, and that these changes could have an impact on viral fitness. Therefore, the dynamics of evolving viral populations in the context of heterotypic antibodies could be related to the increasing clinical severity observed during dengue epidemics.
Key immunological factors determining DENV clinical outcome
We have published two important articles showing cross-neutralization against all four dengue serotypes. The findings could lead to new type of dengue vaccine with one antigen against all four dengue serotypes. In addition, these antigens can be used as biomarkers for protective immunity against dengue. We used the same approach to screen our human monoclonal antibodies collection. We identified both cross neutralizing and enhancement antibodies. In addition, in collaboration with partners in WP8, we identified that T cells were highly activated in asymptomatic viremic individuals while plasma blasts were increasingly developed in dengue patients. These findings provide a new concept for dengue vaccine development.
Role of antigen presenting cells, T cells and metabolic pathway in DENV infection outcome; Risk of dengue in European population
The whole genome expression analysis suggested higher antigen presentation activity and activation of T cells in asymptomatic viremic individuals. It is interesting to note that while several immune systems are higher activated in asymptomatic DENV infected individuals, the pathways, which controlled over-reactive immunity were also highly activated. This finding suggested a balance in immune activation is important in asymptomatic infection and support over reactive immunity underlying pathogenesis of dengue diseases.
Genome wide study of dengue patients from Cuba and Thailand identified new interesting distinct pathways involved in pathogenesis of classical dengue and severe dengue. While confirming a role the phospholipase C pathway in severe dengue as previously reported in the Vietnamese genome wide study, we identified genes in xenobiotic signaling pathway involved in pathogenesis of classical dengue. We calculated the genetic risk using the newly and confirmed susceptible/resistant haplotypes. We showed that European populations (as well as South Asian and USA) present an even higher risk than Southeast Asian populations for severe dengue, while they are the best protected ones against classical dengue.

Potential Impact:
Major results achieved:
Dengue transmission dynamics and dengue control strategies
We generated several results that have potential impact on vector surveillance and dengue control strategies including
1) Individuals who are infected but either pre-symptomatic or asymptomatic are just as infectious to mosquitoes as individuals who have symptoms.
2) A significant proportion of the force of infection of dengue was not occurring in and around the house of an infected individual.
3) Development of an online climate skill interactive tool
4) Provision of all climate information and datasets for the epidemiological modeling.
5) Development of a three module virtual environment simulation system.
6) Development, implementation and validation of an Optimal Control Theory
7) Successful implementation of innovative mosquito control strategy
8) Point of care DENV diagnostic test
Host, viral and vector interactions
We identified several new human and viral factors, which could protect and enhance dengue disease severity and dengue transmission.
9) The demonstration that European Aedes albopictus mosquitoes are as competent for Thai and imported dengue viruses
10) Artificial selection revealed the rapid spread of a single nucleotide variant.
11) Identification and structural characterization of highly potent broadly neutralizing anti-DENV antibodies
12) Highly activated T cell response in asymptomatic DENV infection
13) Cross-enhancement and neutralization of anti-DENV antibodies against Zika virus (ZIKV)
We emphasize the importance of community involvement in research and control of dengue especially in the light of asymptomatic DENV infections. Our findings on asymptomatic DENV infections change the current paradigm of dengue transmission and vaccine development, which hitherto focused on hospitalized dengue patients. Our results have significant impact on future dengue research activities, dengue surveillance and control.

List of Websites:


Sophie Ablott, (Head Of Grant Office)
Tel.: +33 1 45 68 88 47
Fax: +33 1 40 61 39 40


Life Sciences
Record Number: 191900 / Last updated on: 2016-11-21
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