Dissecting global protective immune response to dengue virus at a single-cell resolution
Lead Research Organisation:
Wellcome Sanger Institute
Department Name: Cellular Genetics
Abstract
Dengue virus (DENV) infection is a mosquito-borne disease that infects 390 million people each year. Despite a sharp rise in the number of infected case over the past decade, there is currently no available specific treatment. A vaccine has been developed but is of limited efficacy and has been shown to cause severe side-effects when given to young children. It has been observed that some individuals who are infected with the virus either develop no or very mild symptoms and clear the virus, whereas other individuals develop fever and severe symptoms such as bleeding and shock. Here we aim to compare infected individuals with and without these harmful symptoms to better understand which parts of the immune systems are responsible for clearing the virus and which parts are responsible for causing the often very severe symptoms without clearing the virus effectively.
Human "innate" immune response reacts to general danger signals such as those generated during a viral infection, whereas the "adaptive" immune response recognises the shape of an invader and mount a specific response against this. B and T lymphocytes are the mediators of the adaptive immune response. Each individual T and B lymphocyte carries a unique receptor that is able to recognise a different shape on the infectious agent. Therefore, ideally, immune responses need to be studied at the level of the single cell.
At the Wellcome Trust Sanger Institute (WTSI UK), cutting-edge technology has recently been developed that is able to profile immune cells at the single cell level. Complex "transcriptomics" data are generated, and computational tools for the successful analysis of this type of large-scale data have only recently been developed.
We now propose to apply single cell profiling techniques to one of the most comprehensive Dengue patient cohort assembled in Thailand, one of the main hubs of dengue cases By identifying specific differences in the immune response of individuals with and without disease, we will (1) identify the specific cell subtypes of both the innate and adaptive immune response that is responsible for clearing the virus, (2) provide a molecular description of the receptors in B and T cells that are able to bind the virus. The knowledge gained will help to develop more effective vaccines and anti-viral treatments. In addition, the project will allow Mahidol University (MU, Thailand) to develop a single-cell analysis platform that can be applied to study other infectious diseases in the future.
Human "innate" immune response reacts to general danger signals such as those generated during a viral infection, whereas the "adaptive" immune response recognises the shape of an invader and mount a specific response against this. B and T lymphocytes are the mediators of the adaptive immune response. Each individual T and B lymphocyte carries a unique receptor that is able to recognise a different shape on the infectious agent. Therefore, ideally, immune responses need to be studied at the level of the single cell.
At the Wellcome Trust Sanger Institute (WTSI UK), cutting-edge technology has recently been developed that is able to profile immune cells at the single cell level. Complex "transcriptomics" data are generated, and computational tools for the successful analysis of this type of large-scale data have only recently been developed.
We now propose to apply single cell profiling techniques to one of the most comprehensive Dengue patient cohort assembled in Thailand, one of the main hubs of dengue cases By identifying specific differences in the immune response of individuals with and without disease, we will (1) identify the specific cell subtypes of both the innate and adaptive immune response that is responsible for clearing the virus, (2) provide a molecular description of the receptors in B and T cells that are able to bind the virus. The knowledge gained will help to develop more effective vaccines and anti-viral treatments. In addition, the project will allow Mahidol University (MU, Thailand) to develop a single-cell analysis platform that can be applied to study other infectious diseases in the future.
Technical Summary
Dengue virus (DENV) infects 390 million people each year, mostly in the tropics, with endemic regions expanding rapidly. As yet no effective vaccine or antiviral therapy exists. Here we propose a comprehensive study of the immune responses in asymptomatic vs symptomatic DENV-infected individuals at the level of single cells, as the basis for improvements in vaccine design and better treatment strategies. Recently published data using a bulk microarray approach showed differences in the immune response in DENV-infected individuals with and without symptoms. However, bulk gene expression data has limited resolution. In contrast, single cell analysis generates information on the specific subtypes of immune cells that are activated and that can clear the virus. Utilizing a Dengue patient cohort at Mahidol University, Thailand and cutting-edge single-cell technology at WTSI, we propose to dissect the global protective immune response to DENV infection. Single cell transcriptomes will be obtained from pre-collected peripheral blood mononuclear cells (PBMCs) from asymptomatic and symptomatic DENV infected individuals to identify factors associated with protection versus pathogenesis. Innate immune responses will be assessed by single-cell analysis of T and B cell-depleted PBMCs using an emulsion-based single-cell technology (10x sequencing). In addition, we will perform an in-depth analysis of activated and DENV-specific T cells and activated B cells using the SMART-seq2 protocol, which allows the analysis of TCR and BCR gene segment usage. In cell sub-populations linked to viral clearance, we will identify differentially regulated markers and perform validation experiments to confirm the identified severity-associated factors at the protein level by means of surface marker expression analysis by FACS and cytokine array studies on patient plasma. This will provide crucial information on desirable type immune responses against DENV, paving a way for future vaccine design
Planned Impact
Our comprehensive analysis at the single-cell level of how the immune system of some individuals can provide a protective response to dengue virus infection, while in others it leads to severe disease outcomes, has potential impacts in several areas.
The novel findings from the use of cutting-edge technologies to analyze invaluable samples from patients with different disease outcomes will lead to major scientific advances in our understanding of protective immune responses to this important viral infection that affects a large number of people worldwide. The research community and pharmaceutical companies will benefit from these basic insights to translate them into diagnostic/prognostic tool development, better vaccine designs, and specific antiviral therapies. All these areas are underserved at the moment and are thus in great demand in the combat against dengue virus infection.
As a vector-borne disease carried by the Aedes mosquitoes, the infection affects mainly tropical countries, including Thailand, and the endemic areas are expanding to other parts of the world. Thus, novel diagnostic tools, effective vaccines and therapeutic modalities will eventually be beneficial to a large number of people at risk of the infection. This will also lead to an enormous relief from the economic burden of the disease in developing countries and contribute to a healthier population and workforce.
At the same time, the transfer of the advanced single cell genomics technologies, including computational analysis methods from the UK to Thailand, will provide a great opportunity for Thai researchers to employ this novel technology in other research areas of importance to the country. Thus, this will benefit the Thai research community and biotechnology/pharmaceutical industry as a whole and contribute to economic competitiveness of the country. The young scientists who will be trained in this program will become a key group in exploiting this technology and will help in the training other reseachers from both the academic and industrial sectors in Thailand
The novel findings from the use of cutting-edge technologies to analyze invaluable samples from patients with different disease outcomes will lead to major scientific advances in our understanding of protective immune responses to this important viral infection that affects a large number of people worldwide. The research community and pharmaceutical companies will benefit from these basic insights to translate them into diagnostic/prognostic tool development, better vaccine designs, and specific antiviral therapies. All these areas are underserved at the moment and are thus in great demand in the combat against dengue virus infection.
As a vector-borne disease carried by the Aedes mosquitoes, the infection affects mainly tropical countries, including Thailand, and the endemic areas are expanding to other parts of the world. Thus, novel diagnostic tools, effective vaccines and therapeutic modalities will eventually be beneficial to a large number of people at risk of the infection. This will also lead to an enormous relief from the economic burden of the disease in developing countries and contribute to a healthier population and workforce.
At the same time, the transfer of the advanced single cell genomics technologies, including computational analysis methods from the UK to Thailand, will provide a great opportunity for Thai researchers to employ this novel technology in other research areas of importance to the country. Thus, this will benefit the Thai research community and biotechnology/pharmaceutical industry as a whole and contribute to economic competitiveness of the country. The young scientists who will be trained in this program will become a key group in exploiting this technology and will help in the training other reseachers from both the academic and industrial sectors in Thailand
Publications
| Description | We generated single-cell gene expression and immune receptor expression data from peripheral blood mononuclear cells (PBMCs) acquired from 24 dengue-infected donors with asymptomatic disease, "moderate" dengue fever (DF), and severe dengue hemorrhagic fever (DHF) in Thailand. For the samples from symptomatic donors, the collection time was at one day before defervescence, which was previously shown to manifest active systemic immune activities. To control for the lack of collection time reference in asymptomatic donors, we measured viremia in the samples and only those with similar levels of viremia were used in the experiment. In addition, we produced data from 12 longitudinal samples from 6 symptomatic donors at the acute and convalescence periods to gain more insights into immune dynamics over the course of the disease. In the first cohort, we acquired high-quality single-cell profiles from 134,359 cells following doublet removal and quality control. Cell type annotation was performed with Seurat v4 multimodal reference mapping using a recently described CITE-seq reference of 162,000 PBMCs measured with 228 antibodies. We settled on a granularity of 30 cell types annotated due to an appropriate degree of confidence based on previous knowledge of their biological functions at such resolution. The cell type abundance readily revealed CD8 TEM cells and NK cells being significantly enriched in asymptomatic samples, while plasmablasts/plasma cells being significantly enriched in symptomatic ones. Cell type-specific differential gene expression analysis showed increased expression in peptide biosynthetic process, translational initiation, and viral protein expression in multiple cell types in asymptomatic samples, including memory B cells and CD16 monocytes, which were previously shown to be viral target cells. Overall, the differential gene expression analysis elucidates potentially different mechanisms of viral pathogenesis that might account for better viral control with minimal immunopathology in asymptomatic individuals versus inflammatory phenotype in symptomatic individuals previously known to be enhanced by heterologous antibodies. To further investigate CD8 T cell response, we leveraged the TCR information we generated from the same samples and found that the cells in the CD8 TEM cluster have the highest degree of clonal expansion, indicating that active CD8 effector cells are also in this cluster. Collectively, the data suggest that strong effector CD8 T cell responses are associated with asymptomatic dengue infection, implying that they could be protective in this context. Moreover, we found NK cells, which were shown to be more abundant in asymptomatic donors, to possess different gene expression profiles in different severity groups. |
| Exploitation Route | The in-depth single-cell profiling of systemic immune responses to dengue infection has revealed underlying immune phenotype potentially conferring protection versus immunopathology specific to the virus, which could be translated for better clinical diagnosis and intervention. For example, the association between effector CD8 T cell responses and asymptomatic infection implies that vaccine design, which has been traditionally optimized for strong antibody responses, could be geared more towards CD8 T cell responses as well (for example, with an appropriate adjuvant) to potentially increase vaccine efficacy and minimize side effects due to antibody-dependent enhancement (ADE). Conversely, cell types and/or gene pathways associated with severe diseases identified here could be subjected to further investigations as therapeutic targets. Such scientific and clinical impacts could expectedly be leveraged by both public and private sectors. |
| Sectors | Healthcare,Pharmaceuticals and Medical Biotechnology |
| Description | The grant allowed us to elucidate key similarities and differences in dengue infection with other viral infections, including COVID-19, emphasizing the need for tailored strategies for specific viruses. As the recent global viral pandemic has exemplified why the basic understanding of anti-viral immunity is important for better preparedness and control, it also highlights the impetus to better scientifically address the relatively more neglected viral infection diseases like dengue infection, which was shown to annually affect 390 million people, mostly in developing countries with crippling economic and social consequences. |
| First Year Of Impact | 2021 |
| Sector | Healthcare,Pharmaceuticals and Medical Biotechnology |
| Impact Types | Societal,Economic |
| Description | Dengue Virus |
| Organisation | Mahidol University |
| Country | Thailand |
| Sector | Academic/University |
| PI Contribution | This collaborative project aims to study the global immune responses to DV infection in PBMC, and link TCR and BCR sequences with transcriptional profiles of individual T and B cells in human samples. |
| Collaborator Contribution | Mahidol University have collected and processed the samples prior to sending to Sanger. Researchers have collaborated on data analysis and research output. Oxford University are providing expertise on the purification of the immune cells and providing specific reagents. |
| Impact | Multidisciplinary expertise from leading researchers in different fields to uncover how human immune system response to dengue virus (DENV) infection using a new technology called single-cell RNA sequencing. We are putting together the single-cell genomics expertise of Dr Sarah A Teichmann's lab at the Wellcome Sanger Institute, UK, with the tetramer technology provided by Dr. Juthathip Mongkolsapaya group at Univeristy of Oxford, UK, and apply these techniques to a unique dengue patient cohort that has been assembled by Dr. Ponpan Matangkasombut Choopong (PMC) at Mahidol University (MU), Thailand. |
| Start Year | 2018 |
| Description | Dengue Virus |
| Organisation | University of Oxford |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | This collaborative project aims to study the global immune responses to DV infection in PBMC, and link TCR and BCR sequences with transcriptional profiles of individual T and B cells in human samples. |
| Collaborator Contribution | Mahidol University have collected and processed the samples prior to sending to Sanger. Researchers have collaborated on data analysis and research output. Oxford University are providing expertise on the purification of the immune cells and providing specific reagents. |
| Impact | Multidisciplinary expertise from leading researchers in different fields to uncover how human immune system response to dengue virus (DENV) infection using a new technology called single-cell RNA sequencing. We are putting together the single-cell genomics expertise of Dr Sarah A Teichmann's lab at the Wellcome Sanger Institute, UK, with the tetramer technology provided by Dr. Juthathip Mongkolsapaya group at Univeristy of Oxford, UK, and apply these techniques to a unique dengue patient cohort that has been assembled by Dr. Ponpan Matangkasombut Choopong (PMC) at Mahidol University (MU), Thailand. |
| Start Year | 2018 |