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

Lead Research Organisation: University of Bristol
Department Name: Cellular and Molecular Medicine

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

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Description The overall objective of this collaboration between academic institutes in the Philippines and the UK was to create a state-of-the-art infrastructure to allow better diagnosis of patients who develop severe disease after infection with DENV.

In order to achieve our objectives we analysed samples either collected from patients with different grades of dengue disease severity in the Philippines which were either available in an existing "retrospective" biobank or collected during the course of the project. Thus the project had two phases.

In the first phase of the project samples were analysed by metagenomics and high-throughput proteomic analysis. The metagenomic analysis a) generated >70 full-length DENV sequences, b) identified Chikungunya virus and co-infections, both between different DENV serotypes, and DENV/HIV c) identified a higher prevalence of DENV-4 in Manila than expected and d) provided phylogenetic data showing similarity between Philippine and other south-east Asian isolates. The proteomic analysis of 50 matched serum samples identified a) ~1250 proteins with high confidence, including 125 and 40 proteins that significantly increased or decreased respectively in DENV patients compared to healthy controls and b) a preliminary set of proteins that significantly increased/decreased depending on disease severity.

Importantly, analysis of the pre-existing samples facilitated the establishment of not only experimental but also bioinformatic pipelines for a) metagenomics using next generation sequencing (NGS) to determine the circulating DENV serotypes and b) unbiased proteomic screening to identify biomarkers that discriminate between distinct disease states in DENV infection. The metagenomics analysis methods and bioinformatic pipelines were transferred to the Philippines through two capacity building workshops held in the Philippines by researchers at the Centre of Virus Research (CVR), Glasgow and have now been adopted at the Philippines Genome Centre (PGC).

Whilst the first phase of analysis was ongoing a second (prospective) biobank of longitudinally-collected samples from hospitalised patients throughout the course of dengue disease was established in the Philippines. This database contained detailed clinical data to accompany the specimens and was used in the second phase of the project.

In the second phase of the project we used metagenomic, transcriptomic and high-throughput proteomic analysis to analyse the prospectively collected biobank samples. A total of 107 samples were used for metagenomic analysis using the methods established in the first phase of the project. The data facilitated further detailed phylogenetic analysis of circulating dengue virus strains in the Philippines and identified potential hotspots in the viral genome which may be important in immune evasion. Transcriptomic analysis was done using RNA from 47 patients which allowed us to discriminate patients with different disease severities by transcriptomic profiles. In a deeper analysis we are focussing on using machine learning applications in order to generate algorithms that may identify gene sets predictive of the clinical outcome of DENV infection. In the proteomic analysis 110 patient samples were analysed. Bespoke bioinformatic pipelines were developed to integrate the clinical, proteomic and transcriptomic data. Analysis of the "prospective" proteomic samples led to the total identification of >1800 proteins and a panel of seventy proteins that were able to discriminate different grades of dengue disease severity. The panel of prospective protein biomarkers is being further refined to produce a smaller set (~5-10 proteins) that can be subjected to further validation in the Philippines using a larger patient cohort to develop an immunological test that can be used to predict patients at risk of progressing to severe dengue disease.

During the project two capacity building workshops and five academic exchange workshops were held to transfer capacity to the Philippine partners. This led to training of Filipino scientists in NGS experimental and bioinformatic methods and adoption of these methods at the PGC, a National Facility. The UK partners also had input into the establishment of a proteomics facility at the PGC, advising on overall strategy and equipment choice.

Although the project has ended, the UK and Philippine partners have a very strong commitment to collaborate further on analysis of the large datasets generated during the project to develop diagnostic tests to predict dengue disease severity and continue to build capacity for bioinformatic analysis in the Philippines. This has also led to two Philippines students undertaking PhD degrees at the University of Glasgow and two Philippines students undertaking MSc degrees at the University of Bristol.
Exploitation Route Expertise in next-generation sequencing methods and bioinformatic analysis has already been transferred to scientists at the Philippine Genome Center (PGC) by the UK partners. Further exchange is ongoing to continue to build expertise at the PGC, especially in the training of younger researchers in bioinformatic analysis, that can be applied not only to virology but also health sciences in the Philippines.

Analysis of the large transcriptomic and proteomic data sets generated in the project is still ongoing by the UK and Filipino partners in order to identify specific RNA transcripts and proteins that can be used to develop diagnostic tests for dengue disease severity. The UK and Philippine partners are validating candidate biomarkers to produce a smaller set of refined RNA and protein biomarkers that can be patented for use as a diagnostic test.
Sectors Digital/Communication/Information Technologies (including Software),Education,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description MRC UK-Indonesia Newton Fund
Amount £570,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start  
 
Description Newton Advanced Fellowship
Amount £110,000 (GBP)
Organisation Academy of Medical Sciences (AMS) 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2016 
End 02/2019
 
Description Newton MRC-NSTDA UK- Thai fund
Amount £714,015 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 04/2018 
End 03/2021
 
Description Proteomic identification of serum biomarkers for Dengue
Amount £19,450 (GBP)
Organisation University of Bristol 
Sector Academic/University
Country United Kingdom
Start 05/2018 
End 07/2018
 
Title Improvements to metagenomic studies on dengue virus 
Description We have developed methods for next generation sequencing of clinical samples from dengue virus-infected patients that has enabled a) bioinformatic approaches to assemble almost complete contigs of all 4 dengue serotypes, b) minor variants in dengue viral sequences that may be linked to the host response and c) identification of viruses other than dengue in clinical samples. 
Type Of Material Biological samples 
Year Produced 2018 
Provided To Others? No  
Impact The studies represent the starting point for a Virome database of viral sequences that circulate in the Philippines. 
 
Title Metagenomic analysis of human samples infected with dengue virus 
Description A pipeline has been developed for determining the presence of dengue virus and other pathogens in clinical samples from the Philippines. The pipeline has enabled the detection of different serotypes of dengue virus as well as the presence of other viruses (chikungunya and HIV). 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? Yes  
Impact No notable impact as yet but the analytical method will be assessed in all collaborating centres in the study. 
 
Title Proteomic bioinformatic pipeline 
Description A pipeline has been developed for analysing proteomic data produced from mass spectrometric analysis of serum samples from patients with different grades of dengue disease severity in the Philippines. The pipeline has enabled the detection of proteins that differ in abundance in patient serum. 
Type Of Material Improvements to research infrastructure 
Year Produced 2018 
Provided To Others? No  
Impact none as yet 
 
Title Dengue-GLUE 
Description Using the GLUE framework developed at the CVR, a test version of a Dengue-GLUE database is being developed which, in the long-term, will be deposited with collaborators in the Philippines as part of the legacy of the study. 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact None as yet 
 
Title Proteomic database for selection of candidate biomarkers 
Description A proteomic database containing the proteomic profile of 160 serum samples fro dengue infected patients has been provided to the Philippines researchers. A panel of 70 potential biomarkers has been selected for further validation by targeted proteomics. The proteomic databases contains proteomic, transcriptomic and clinical data. 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? Yes  
Impact The data bases has been used to select potential biomarkers for further validation prior to patenting of selected biomarkers. 
 
Description A Cross-Sectional on the Seroprevalence of Zika Virus Infection Among Filipino Children with Isolated Microcephaly 
Organisation University of Philippines Manila
Country Philippines 
Sector Academic/University 
PI Contribution The UK partners will provide the reagents needed for the virus infectivity assays and the expertise to train Filipino scientists on growth and assay of Zika virus.
Collaborator Contribution The Filipino partners wil determine the: - seroprevalence of ZIKV infection among Filipino children with isolated microcephaly - seroprevalence of maternal ZIKV infection among Filipino children with isolated microcephaly - sociodemographic and medical factors associated with ZIKV infection among Filipino children with isolated microcephaly
Impact Grant proposal submitted for funding in the Philippines. Currently under revision for funding.
Start Year 2016
 
Description UK-Indonesian Consortium to Identify Biomarkers Predictive of Dengue Disease Severity 
Organisation Universitas Indonesia
Country Indonesia 
Sector Academic/University 
PI Contribution The research team at the University of Bristol will use an integrated transcriptomic and proteomic approach to analyse the serum from Indonesian patients with differing grades of disease severity to identify prognostic biomarkers to predict dengue disease severity.
Collaborator Contribution The partners in Indonesia will provide patient samples from cohorts of patients with differing degrees of dengue disease severity. The partners at the University of Glasgow will undertake metagenomic and transcriptomic analysis of the samples. In combination with the Bristol researchers they will undertake an integrated analysis of the omic datasets to indentify biomarkers for dengue disease severity.
Impact Funded grant: Source: MRC-Newton - Indonesia Title: UK-Indonesian Consortium to Identify Biomarkers Predictive of Dengue Disease Severity Amount of the Grant: £ 589,531 Start Date and Duration: Nov, 2017 - Oct, 2019; 24 months
Start Year 2017
 
Description UK-Indonesian Consortium to Identify Biomarkers Predictive of Dengue Disease Severity 
Organisation University of Glasgow
Department MRC - University of Glasgow Centre for Virus Research
Country United Kingdom 
Sector Academic/University 
PI Contribution The research team at the University of Bristol will use an integrated transcriptomic and proteomic approach to analyse the serum from Indonesian patients with differing grades of disease severity to identify prognostic biomarkers to predict dengue disease severity.
Collaborator Contribution The partners in Indonesia will provide patient samples from cohorts of patients with differing degrees of dengue disease severity. The partners at the University of Glasgow will undertake metagenomic and transcriptomic analysis of the samples. In combination with the Bristol researchers they will undertake an integrated analysis of the omic datasets to indentify biomarkers for dengue disease severity.
Impact Funded grant: Source: MRC-Newton - Indonesia Title: UK-Indonesian Consortium to Identify Biomarkers Predictive of Dengue Disease Severity Amount of the Grant: £ 589,531 Start Date and Duration: Nov, 2017 - Oct, 2019; 24 months
Start Year 2017
 
Description National Research Council of the Philippines (NRCP) - Division of Medical Sciences Presentations 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The two UK PIs on the grant gave presentations to the National Research Council of the Philippines (NRCP) - Division of Medical Sciences audience at The Medical City (Ortigas City, Manila) on the 09/08/2016. The talks were designed to engage medical practitioners with basic science. Subsequently two Filipino clinicians have either enrolled or will enrol in MSc research programmes at the University of Bristol.
Year(s) Of Engagement Activity 2016
URL http://www.nrcp.dost.gov.ph/latest-news/194-nrcp-medsci-group-gets-knowledge-boost-on-latest-virus-r...
 
Description Next Generation Workshop - Philippines Genome Center 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact A workshop dedicated to next generation sequencing of viruses was developed and presented in the Philippines to build capacity in the country for viral genomics.
Year(s) Of Engagement Activity 2017
 
Description Singapore-Malaysia visit March 2019 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact I attended an eight day school recruitment roadshow in Singapore and Malaysia presenting talks at local schools to final and pre-final year students. I integrated the results of the project and the idea of big data analysis into the talks. The talk was repeated to nine schools to audiences ranging from 10 - > 100 students. The talk encouraged many questions on the use of 'omics approaches to fight infectious disease and big data analysis.
Year(s) Of Engagement Activity 2019
 
Description UK-Indonesia Infectious Diseases Workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Workshop to foster collaborative research between UK and Indonesian scientists.
Year(s) Of Engagement Activity 2016
 
Description Viral Bioinformatics - Philippines Genome Center 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact A workshop dedicated to bioinformatics for analysis of viral sequence data was developed and presented in the Philippines to build capacity in the country for viral genomics.
Year(s) Of Engagement Activity 2017