Periodic Reporting for period 2 - CoCID (Compact Cell-Imaging Device to provide insight into the cellular origins of diseases and to aid in the development of novel therapeutics)
Reporting period: 2022-07-01 to 2023-12-31
Viral diseases are an important cause of mortality and also carry a significant social and economic cost to humanity. For example, the cost of influenza virus care in 2018 was estimated at about €29 billion, or 2% of healthcare costs of the EU. Other viruses, such as hepatitis C virus (HCV), with incidence of 8.7 per 100 000 people in EU, can cause a lifelong infection and is a major cause of liver cancer. The SARS-CoV-2 global pandemic will probably lead to 50-fold or more increase in flu healthcare costs in Europe. Some viruses, such as herpes simplex virus 1 (HSV-1), can be used to kill cancer cells. Overall, viruses have an important impact on human health and the costs of development of antiviral therapies and virus mediated cancer therapy are considerable, in terms of economic, physical, social and mental well-being.
Analysis of 3D biological cell samples is critical for understanding the mechanisms of viral disease and for development of novel therapeutics. Soft X-ray microscopy (SXM) is the unique technology that can image whole intact cells in 3D under normal and pathological conditions without labelling or fixation, at high throughput and spatial resolution. The main challenge of SXM is that the photonic illumination required for imaging is currently only available at synchrotrons and only a very small fraction of the infectious disease research community has been able to access this imaging modality.
The European Union Horizon 2020 project called CoCID, the ‘Compact Cell Imaging Device’ will address this challenge by developing a lab-scale soft X-ray microscope for 3D imaging of whole cells in the laboratory. The capabilities of this device will be demonstrated through a series of virology use cases to generate new scientific knowledge on the viral life cycle and host cell response to viral infection.
Analysis of 3D biological cell samples is critical for understanding the mechanisms of viral disease and for development of novel therapeutics. Soft X-ray microscopy (SXM) is the unique technology that can image whole intact cells in 3D under normal and pathological conditions without labelling or fixation, at high throughput and spatial resolution. The main challenge of SXM is that the photonic illumination required for imaging is currently only available at synchrotrons and only a very small fraction of the infectious disease research community has been able to access this imaging modality.
The European Union Horizon 2020 project called CoCID, the ‘Compact Cell Imaging Device’ will address this challenge by developing a lab-scale soft X-ray microscope for 3D imaging of whole cells in the laboratory. The capabilities of this device will be demonstrated through a series of virology use cases to generate new scientific knowledge on the viral life cycle and host cell response to viral infection.
Technology Objectives
A fluorescent microscope has been integrated into the sample chamber of the SXM and is now routinely used as part of the SXT-100 imaging workflow. A dual capillary/grid sample holder docked to the existing cryo stage will be implemented in Q1 2023. A basic data pipeline for grid-based datasets is in place that allows image registration, alignment and reconstruction of the SXT-100 datasets. A faster automated pipeline will be ready by the end of 2022.
Biological Objectives
1.To investigate reversion of hepatitis C virus-induced morphological alterations by antiviral drugs
We have assessed kinetics of the reduction of viral antigen and genomic RNA levels after effective antiviral treatment of replicon cells using immunofluorescence microscopy, Western-Blot as well as quantitative PCR. We have also established a correlation between virus elimination and restoration of normal cell homeostasis.
2.To understanding cross-species transmission mechanisms of hepatitis E virus
Microscopy workflows and many biological tools have been established and optimised. Structural effects of HEV Genotype 1 replicons in human cells have been analysed by electron microscopy and correlated low resolution light microscopy. Putative structural changes during infection have been seen and are ready to be further investigated and quantified using SXM on a whole cell scale.
3.To study SARS-Cov-2 virus-induced remodelling of the host cell
Cell culture work has established the best conditions to maintain virus stocks and to find suitable cell models to enable the study SARS-CoV2 by SXM as well as other techniques. UKHD and others were able to address the major points of T8.1 i.e. the characterization of the changes to overall cell architecture of lung epithelial cells Calu3 upon SARS-CoV2 infection, using plastic room temperature FIB-SEM and TEM tomography.
4. To elucidate the herpesvirus-induced chromatin reorganization
SXM imaging combined with other imaging techniques and advanced data analysis revealed that the spatial organization and density of chromatin change dramatically in the late stage of infection (D9.1). Viral infection induces a stress condition in the host cells, which causes a dramatic change in the morphology and biological function of mitochondria. SXM developed in the CoCID project allows detailed analysis of structural changes of mitochondria.
A fluorescent microscope has been integrated into the sample chamber of the SXM and is now routinely used as part of the SXT-100 imaging workflow. A dual capillary/grid sample holder docked to the existing cryo stage will be implemented in Q1 2023. A basic data pipeline for grid-based datasets is in place that allows image registration, alignment and reconstruction of the SXT-100 datasets. A faster automated pipeline will be ready by the end of 2022.
Biological Objectives
1.To investigate reversion of hepatitis C virus-induced morphological alterations by antiviral drugs
We have assessed kinetics of the reduction of viral antigen and genomic RNA levels after effective antiviral treatment of replicon cells using immunofluorescence microscopy, Western-Blot as well as quantitative PCR. We have also established a correlation between virus elimination and restoration of normal cell homeostasis.
2.To understanding cross-species transmission mechanisms of hepatitis E virus
Microscopy workflows and many biological tools have been established and optimised. Structural effects of HEV Genotype 1 replicons in human cells have been analysed by electron microscopy and correlated low resolution light microscopy. Putative structural changes during infection have been seen and are ready to be further investigated and quantified using SXM on a whole cell scale.
3.To study SARS-Cov-2 virus-induced remodelling of the host cell
Cell culture work has established the best conditions to maintain virus stocks and to find suitable cell models to enable the study SARS-CoV2 by SXM as well as other techniques. UKHD and others were able to address the major points of T8.1 i.e. the characterization of the changes to overall cell architecture of lung epithelial cells Calu3 upon SARS-CoV2 infection, using plastic room temperature FIB-SEM and TEM tomography.
4. To elucidate the herpesvirus-induced chromatin reorganization
SXM imaging combined with other imaging techniques and advanced data analysis revealed that the spatial organization and density of chromatin change dramatically in the late stage of infection (D9.1). Viral infection induces a stress condition in the host cells, which causes a dramatic change in the morphology and biological function of mitochondria. SXM developed in the CoCID project allows detailed analysis of structural changes of mitochondria.
Progress beyond the state of the art
1. Bring synchrotron soft X-ray microscopy capabilities to the labs
2. Provide unique flexible sample presenting systems
3. Establish integrated multiscale hybrid microscopy methods
The SXT-100 is now fully operational and routinely imaging cryo prepared mammalian cells for CoCID partners and other groups. The implementation of the in-line fluorescent microscope achieved during RP1 has been a very significant development in allowing fast screening and identification of cells for SXM imaging. Innovations 2 & 3 will be addressed during RP2, with the development of a capillary sample mounting mechanism and correlative (SXM-EM-FM) microscopy workflows.
Expected results
1. 3D assessment of HCV-induced changes in mitochondria and endoplasmic reticulum (ER) in native, cryo preserved infected cells, by imaging with SXM, will be used for development and testing of antiviral drugs.
2. SXM of human polarized and non-polarized cell lines and tissues will allow detailed analysis of HEV replication and assembly in the presence or absence of antiviral drugs.
3. SXM will allow spatiotemporal quantitative analysis of SARS-CoV-2 and Dengue virus induced modification of cytoplasmic organelles essential for understanding the underlying mechanisms of infection and for design of antiviral drugs.
4. 3D SXM analyses of mitochondria and chromatin distribution, compaction, and organization will allow unique detailed analysis of HSV-1 -induced changes for development of effective HSV-1 vectors for oncolytic viral therapies.
Potential impacts
1. Making an extremely valuable imaging modality, that can help accelerate the understanding of the cellular origin of disease, accessible to the whole market.
2. Ability to image an intact cell allows changes in large subcellular structures that are linked to disease, to be studied.
3.Natural contrast gives the sensitivity and integrity needed to study cell structure without having to stain the cell with metals.
4.Linear relationship between absorption and density gives increased specificity and allows segmentation of organelle in cell.
5. Complementary nature of cryo-SXM and cryo-TEM allows end users to combine benefits of both modalities.
6. Imaging the time-sequenced reversal of HCV-induced cell changes allows researchers understand antiviral drug impact and can help adapt these to new viruses.
7. Imaging effect of antiviral drugs on HEV in humans and pigs will increase understanding of replication modes and help accelerate development of vaccines.
8. Understanding the replication steps of SARS-CoV2 in a cell will help accelerate development of antiviral drugs and vaccines.
9. Correlate chromatin structure changes with effects of HSV infection to help find HSV vaccine for virus that effects 4 billion people.
10. Strengthen EU position in the market for research microscopes.
11.Enhance innovation capacity leading to accelerated understanding of disease origins.
12. Creating new insights into disease causation accelerates progress in the field of drug therapy and vaccine development.
13. Meeting the needs of an unserved market for a lab-scale SXM.
14. Grow SiriusXT as a manufacturer of commercial SXT-100 microscopes.
1. Bring synchrotron soft X-ray microscopy capabilities to the labs
2. Provide unique flexible sample presenting systems
3. Establish integrated multiscale hybrid microscopy methods
The SXT-100 is now fully operational and routinely imaging cryo prepared mammalian cells for CoCID partners and other groups. The implementation of the in-line fluorescent microscope achieved during RP1 has been a very significant development in allowing fast screening and identification of cells for SXM imaging. Innovations 2 & 3 will be addressed during RP2, with the development of a capillary sample mounting mechanism and correlative (SXM-EM-FM) microscopy workflows.
Expected results
1. 3D assessment of HCV-induced changes in mitochondria and endoplasmic reticulum (ER) in native, cryo preserved infected cells, by imaging with SXM, will be used for development and testing of antiviral drugs.
2. SXM of human polarized and non-polarized cell lines and tissues will allow detailed analysis of HEV replication and assembly in the presence or absence of antiviral drugs.
3. SXM will allow spatiotemporal quantitative analysis of SARS-CoV-2 and Dengue virus induced modification of cytoplasmic organelles essential for understanding the underlying mechanisms of infection and for design of antiviral drugs.
4. 3D SXM analyses of mitochondria and chromatin distribution, compaction, and organization will allow unique detailed analysis of HSV-1 -induced changes for development of effective HSV-1 vectors for oncolytic viral therapies.
Potential impacts
1. Making an extremely valuable imaging modality, that can help accelerate the understanding of the cellular origin of disease, accessible to the whole market.
2. Ability to image an intact cell allows changes in large subcellular structures that are linked to disease, to be studied.
3.Natural contrast gives the sensitivity and integrity needed to study cell structure without having to stain the cell with metals.
4.Linear relationship between absorption and density gives increased specificity and allows segmentation of organelle in cell.
5. Complementary nature of cryo-SXM and cryo-TEM allows end users to combine benefits of both modalities.
6. Imaging the time-sequenced reversal of HCV-induced cell changes allows researchers understand antiviral drug impact and can help adapt these to new viruses.
7. Imaging effect of antiviral drugs on HEV in humans and pigs will increase understanding of replication modes and help accelerate development of vaccines.
8. Understanding the replication steps of SARS-CoV2 in a cell will help accelerate development of antiviral drugs and vaccines.
9. Correlate chromatin structure changes with effects of HSV infection to help find HSV vaccine for virus that effects 4 billion people.
10. Strengthen EU position in the market for research microscopes.
11.Enhance innovation capacity leading to accelerated understanding of disease origins.
12. Creating new insights into disease causation accelerates progress in the field of drug therapy and vaccine development.
13. Meeting the needs of an unserved market for a lab-scale SXM.
14. Grow SiriusXT as a manufacturer of commercial SXT-100 microscopes.