Triple-D Targets: The UK-Philippines Dengue Diagnostic and Drug Targets Research Consortium

Dengue is the most important mosquito-borne disease of humans and is constantly spread in the Philippines, which has the 4th largest dengue disease burden of all Southeast Asian countries. Infection with dengue virus (DENV) causes a spectrum of clinical illnesses, ranging from dengue fever, a debilitating but self-limited condition to the more severe and potentially fatal dengue haemorrhagic fever. Dengue has a major impact on the health and economy of the Philippines as epidemics occur every 2-3 years. Over 500 000 confirmed cases occurred from 2008 - 2012 with > 3000 deaths. Although most dengue cases could be managed at home, the lack of a diagnostic test to predict those individuals who progress from mild to severe disease forces patients and healthcare providers to seek hospital admissions for "safety purposes", saturating an already overwhelmed healthcare system.

The overall objective of this collaboration between academic institutes in the Philippines and the UK is to create a state-of-the-art infrastructure that that will allow better diagnosis of patients who develop severe disease after infection with DENV. In order to achieve our objective we will establish a very well characterised biobank of patient samples, collected from patients with different dengue disease outcomes as the disease progresses. We will use sophisticated methods to look at the strains of DENV that are circulating in the Philippines and also identify any changes in patient RNA or proteins which we can detect in blood that might be linked to serious disease associated with DENV infection. The methods will be both high throughput and very sensitive so that even changes in the abundance of low-level transcripts or proteins will be detected. We will also be able to detect the frequency of minor changes in the virus and investigate whether these may be linked to disease. This technology will be transferred to the Philippines so that they can monitor the virus infections, which are circulating in the country.

The results will produce large amounts of data that will be brought together using computational methods, either available or be developed by the Filipino and UK groups. The UK partners have particular experience with the bioinformatics tools needed and will provide these to their Filipino counterparts. These skills and those for detecting mutations in DENV will be transferred to the Philippines through short-term scientific exchange visits, regular real-time cyber-mentoring sessions as well as joint workshops throughout the project.

Computational analysis of the datasets will allow us to identify biomarkers in patients, which will be validated with additional samples. After validation, any biomarkers can then be used to develop diagnostic tests to predict those individuals at risk of progressing to severe disease and to monitor DENV variability and detect other infections associated with febrile disease. We will also identify cell pathways in infected patients which can be targeted with existing drugs, potentially to prevent severe disease. Overall, the project will significantly support the building of research capacity in the Philippines. Critically, the training and technology transfer provided by the UK partners will help to establish an adaptable technological framework that is also relevant to many other infectious disease areas in the Philippines.

We will adopt an integrated 'omics approach, to correlate dengue disease severity with viral sequence variation and changes in the host transcriptome and proteome, using whole blood and serum samples from dengue patients with well-defined clinical outcomes.
Existing serum samples (from the NIH-UPM biobank) from the following 5 patient groups will be selected for viral metagenomic and proteomic analysis a) classical DF, b) DHF, c) DSS, d) acute febrile illness (non-dengue) and e) healthy volunteers (5 of each). Metagenomics will generate viral sequence data for analysis by the UoG's bespoke bioinformatics pipeline. Serum proteins will be identified and quantified by tandem mass tagging of the peptides prepared from serum proteins by tryptic digest followed by high-throughput LC-MS/MS analysis.
A second phase of 'omics analysis will utilise prospectively collected samples. The samples will correspond to the above disease conditions except that multiple patient samples will be collected a) upon enrolment, b) during significant clinical events and c) convalescence. Along with sample storage, clinical variables for patients will also be collected. Further serum samples will be analysed by metagenomics to design virus-specific capture probes for NGS target enrichment. 100 whole blood samples (10 of each dengue condition at 3 times and 10 controls) and corresponding serum samples will be used for RNAseq transcriptomic and proteomic studies respectively.
The clinical and 'omics datasets will be integrated using a bespoke bioinformatics pipeline and software designed by the partners. This will identify candidate mRNA and protein biomarkers which will be validated by qRT-PCR and targeted proteomics using a larger prospective sample collection. Validated biomarkers will form the basis for novel diagnostic tests to predict severe dengue disease. Bioinformatics analysis will also identify host pathways that are potentially druggable, using an in silico modelling approach.

Who will benefit from the research?

A key element of our proposal will be the creation of a collaborative network between Filipino and UK groups that will build mutually beneficial relationships for the future. UoB and UoG will bring a range of state-of-the-art expertise to the Philippines, promoting the use of novel technologies and analytical tools in bioinformatics. This will facilitate translatable applications both in the area of predictive diagnosis and the identification of new targets for dengue therapy. Within such a broad collaborative network, there will be unique opportunities for training young scientists from both countries via exchange visits and cyber networking. This will yield benefits for young Filipino scientists, who will have access and training in the most recent innovations in 'omics technologies and a suite of bioinformatic tools, and to young UK scientists, who will benefit from the opportunity to address the challenges of applying advanced systems in a developing country.

Importantly, this collaborative endeavor will also catalyze and "leapfrog" the level and degree of scientific work within the Philippines on a major disease topic that has financially crippled a majority of the marginalised communities due to the cost of medical care. It will also stimulate economic growth through science and technology by developing relevant products for health and industry. In a wider context, the collaborative interaction between the three groups will advance dengue research in both countries and nurture the slowly growing number of young graduates wishing to pursue a scientific career to supply the much needed critical mass of local scientific experts. The development of new diagnostics and drug candidates will increase the number of technology business incubators in the Philippines and opportunities for scale-up manufacturing and the rise of new leaders in biotechnology in the region.

How will they benefit?

The research plan will be achieved through an integrated strategy involving Filipino and UK researchers. The expertise of the UoB and UoG researchers in high-throughput 'omics along with any integrated bioinformatic analytical tools will be transferred to UPM through short-term scientific exchange visits, regular real-time cyber-mentoring sessions and joint workshops throughout the project. This will significantly support the Philippines' research capacity enhancement program. Critically, the training will help to establish an adaptable technological framework that is relevant to many other infectious disease areas in the Philippines. In this context, the coupling of two major UK centres with complementary strengths and expertise in virus research brings synergistic benefits to UPM and considerably enhances the scope of the project. A major objective of the program is the creation of novel dengue diagnostic platforms that will not only provide the tools for identifying the presence or absence of the disease but more importantly provide clinicians with an objective basis for risk stratifying dengue cases that require hospitalization. The described collaborative research programme between scientists in the Philippines and the UK will hopefully be the start of a long-term relationship that will create a bridge between clinical and basic research, not only focused on dengue but on other biomedical problems of interest to both parties.