Periodic Reporting for period 2 - EVIDENT (Ebola Virus Disease - correlates of protection, determinants of outcome, and clinical management) Reporting period: 2016-05-01 to 2016-10-31 Summary of the context and overall objectives of the project EVIDENT is a Research and Innovation project that focuses on the Ebola virus (EBOV) disease (EVD) epidemic in West Africa. It is closely linked with and builds on the European Mobile Laboratory (EMLab) project, which has deployed three laboratory units to the affected countries in West Africa and performed EVD diagnostics for more than 20,000 samples from 2014 to 2016. EVIDENT has immediate, mid-term and long-term objectives to combat the current and future EVD epidemics.The specific objectives of EVIDENT are:- Providing key information needed to implement efficient convalescent plasma treatment and a toolkit to determine the suitability of plasma for treatment.- Providing key information on immunity of survivors needed to estimate the efficacy of experimental vaccines.- Improving supportive treatment of patients and reducing hospital case fatality rate by providing information on biomarkers and relevant co-infections.- Providing information on pathophysiological changes and immunological determinants to infer new strategies for treatment of EVD.- Monitoring development of mutations in EBOV genomes during the epidemic and enhancing our preparedness to determine the relevance of these changes in experimental systems.- Protecting health care workers and communities by providing information on virus shedding and estimation of infectiousness in various stages of EVD.- Support to operational research projects of other partners in the field, specifically vaccine and drug trails.- Strengthening cooperation of biosafety level 4 (BSL-4) facilities and building a pan-European research area in the field of highly pathogenic viruses.Main conclusions from the work of EVIDENT are:- Convalescent plasma should be selected for treatment according to neutralising antibody titer.- T cell and B cell response are likely correlates of protection in survivors; and vaccines should elicit both. - EVD is characterised by serious disturbance of T cell homeostasis, a pathophysiological feature that might be amenable to therapeutic intervention. - Ebola virus evolves during epidemics with a mutational rate comparable to other RNA viruses; emerging mutations may change the phenotype of the virus. - Real-time molecular epidemiology based on sequencing in the field greatly enhances tracing of transmission chains and should be an integral part of outbreak response in the future. - Ebola virus persists in various body fluids for long periods of time, remains infectious during persistence, and may be transmitted via breastfeeding or sexual contact. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far "EVIDENT has achieved the following results:1. Immune response of survivors: We have assessed the antibody titers, including neutralising antibodies, and T cell reactivity in survivors. The neutralising antibody titers remain stable after discharge for long periods of time (at least a year). Glycoprotein antibodies as measured in ELISA can serve as a surrogate for neutralising activity. As survivors are supposedly protected from re-infection, the observed level of antibody and T cell response should be considered protective. 2. Management of patients: Set up of clinical chemistry measurement in the field revealed frequent renal failure, electrolyte disturbance, and evidence for bacterial super-infection. Main co-infection was malaria, while chronic virus infections were rare. Age, virus load, and co-infection with malaria in children 5-14 years of age are independent predictors of poor outcome. 3. Ebola virus evolution: Our deep sequencing program revealed the evolutionary history and molecular clock of the virus during the epidemic with multiple spreading events of the virus across country borders. Furthermore, we established nanopore sequencing technology in Guinea to follow the molecular epidemiology of the virus in real-time and support field epidemiology and contact tracing efforts. Replicon and reverse genetics systems were established to study the consequences of virus mutations that emerged during the epidemic. 4. Pathophysiology and immunology of EVD: We have measured a wide range of soluble cytokines, chemokines and growth factors, and studied the T, Vδ2T, and NK cell response during acute infection. Fatal and surviving EVD patients showed robust T cell activation consistent with biomarker analysis. However, in fatal patients markers of T cell ‘exhaustion’, in particular CTLA-4 and PD-1, were upregulated demonstrating that defects in the T cell homeostasis are associated with poor outcome of EVD. 5. Virus persistence and shedding: We found that essentially all body fluids contain virus RNA until convalescence. Virus persists after convalescence in breast milk and seminal fluid. Time to clearance of EBOV RNA from seminal fluid is variable and may be >10 months. 6. Exploitation and dissemination: Results of EVIDENT have been presented to national authorities to facilitate outbreak response; to WHO to be considered in guidelines; to pharmaceutical companies to guide in the development of EVD vaccines; and to the wider scientific community at international conferences. The data have been published in high-ranking scientific journals, including ""Nature"". Thus far, 16 articles are published or in press. " Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) The antibody and T cell data from survivors provide key information needed to implement efficient convalescent plasma treatment and to estimate the efficacy of experimental vaccines, and facilitate the licensing of current and future EBOV vaccines.The data on biomarkers, co-infections, and virus load will improve supportive clinical care, provide new prognostic information on the course of the disease, and predict complications, such as liver or renal failure. Some pathologic clinical chemistry parameters can be corrected (such as electrolytes, glucose) even in resource-poor settings. Our results have also implications for antimalaria and antibacterial therapy.Our sequencing data have shown to which extent and in which regions the virus changes during an epidemic; and we have explored the consequences of mutations of concern. The set-up of a sequencing facility in the field demonstrated the great value of combining field and molecular epidemiology in outbreak situations. This concept is a blueprint for future epidemics. The project also has a long-term impact on research and innovation. Studies on pathogenesis of the acute infection, including the pathophysiology of the immune response and analysis of relevant pathways and mediators of disease will facilitate the design of therapeutic strategies interfering with this pathophysiological cascade.Our studies on virus shedding will improve the safety of health care workers and family members, who are in contact with patients in early phase and after discharge. We now know that a large variety of body fluids contains virus and patients are potentially infectious after discharge for a long time. We also observed that sensitive PCR assays might not be able to detect the virus in a few patients at early stage.In conclusion, main socio-economic impact of EVIDENT will result from application of the generated knowledge in the operational response to future epidemics as well as the development of medical countermeasures in the inter-epidemic time. Both will reduce the impact of future epidemics on the affected population and the economy of the affected countries. EVIDENT project logo
