World Hepatitis Day is a movement aimed at raising awareness of the global burden of viral hepatitis and promoting progress in prevention, diagnosis and treatment. It is one of seven global public health days officially mandated by the World Health Organization. This year’s theme, “Hepatitis Can’t Wait”, emphasises the urgency required to address gaps in diagnosis, treatment, vaccination, investment, policy, and social acceptance of people affected by hepatitis A, hepatitis B, hepatitis C, hepatitis D, and hepatitis E.
Given the high prevalence of hepatic diseases and the heavy reliance on invasive liver biopsies for diagnosis, progress in this clinical area is urgently needed. On World Hepatitis Day 2021, we share a selection of studies which have explored the utility of extracellular vesicles (EVs) as diagnostic biomarkers in viral hepatitis and other liver diseases.
Early progress towards EV-based diagnosis of non-alcoholic fatty liver disease and chronic hepatitis C
Paper: Kornek M et al (2012). Circulating microparticles as disease-specific biomarkers of severity of inflammation in patients with hepatitis C or nonalcoholic steatohepatitis. Gastroenterology. 143(2)448-458. https://doi.org/10.1053/j.gastro.2012.04.031
This was a seminal paper showing that quantification of circulating immune cell EVs can be used to assess the extent and characteristics of hepatic inflammation associated with chronic liver disease.
Background: Hepatitis C and non-alcoholic fatty liver disease (NAFLD) are two serious and highly prevalent hepatic diseases. Hepatitis C is an infectious disease caused by the hepatitis C virus and affects approximately 71 million people worldwide.<super-script>1<super-script> Known as a ‘silent disease’, hepatitis C usually remains undetected until significant liver damage has occurred.
NAFLD is comprised of a range of chronic liver conditions characterised by excessive fat in the liver. The disease can present as benign steatosis or may progress to a more serious form: nonalcoholic steatohepatitis (NASH), which is associated with severe inflammation, fibrosis and hepatocyte damage. Currently, liver biopsy is the diagnostic gold standard used for patients suspected of having NASH.<super-script>2<super-script>
Consistent with the knowledge that chronic hepatitis C is associated with significant CD8+ and CD4+ T-cell infiltration, the group had shown previously that circulating MPs derived from CD4+ and CD8+ T cells are elevated in those with chronic hepatitis C, and that these levels are correlated with histological severity.<super-script>3<super-script>
Study approach: Kornek et al. (2012) compared profiles of circulating cell membrane-derived microparticles (MPs) across different groups: healthy volunteers, people with chronic hepatitis C, and people with NAFLD across a range of severity. An extended panel of MP surface markers was studied, including markers from monocytes/macrophages and myeloid dendritic cells, neutrophils, platelets and endothelial cells, and invariant natural killer T cells.
Key findings: Unique differences in MP characteristics were found across the different groups. CD4+ and CD8+ T-cell-derived MPs were elevated in the plasma of patients with chronic hepatitis C, compared to those of healthy individuals. While these MPs could not distinguish groups with chronic hepatitis C vs NAFL/NASH, MPs from CD15+ and CD41+ were significantly lower in those with NAFL/NASH, compared to those with chronic hepatitis C. In line with the knowledge that CD14+ and iNKT cells are central to adipose tissue inflammation, MPs with these markers were characteristically elevated in the NAFL group, and was further increased in those with histologically severe NASH – but not in those with chronic hepatitis C.
Establishing protocols for the removal of virions from EV-containing samples
Paper: Jung S, Jacobs K, Shein M, et al. (2020) Efficient and reproducible depletion of hepatitis B virus from plasma derived extracellular vesicles. Journal of Extracellular Vesicles. 10(2)e12040. https://doi.org/10.1002/jev2.12040
In this technical report, Jung et al. (2020) describe a method for purifying EVs (without bound antibodies) from hepatitis B virus-containing plasma. The method could potentially be used to isolate virus-free EVs from other viral samples.
Background: Hepatitis B remains a global health burden with poorly understood pathophysiology and is a leading cause of liver cancer. While there has been major progress towards eliminating hepatitis B as a public health threat, largely thanks to vaccine development, hepatis B infections affect more than 250 million chronical carriers and accounts for nearly 900,000 deaths each year. As EVs have been implicated in almost all physiological processes, the EV field is keen to explore how EVs contribute to viral infections – including hepatitis B.
The purification of EV-containing samples is an important step in functional studies of EVs and viral infections, as it ensures the particles being measured are actually EVs. To facilitate studies of EVs and their role in viral infections, there is first a need to remove virons and antibody contaminants from EV-containing samples. However, the separation of EVs from virions is challenging as they can overlap in size, density, and membrane composition.
Study approach: Jung et al. established a protocol for EV purification from hepatitis B virus-containing plasma using Izon’s qEVoriginal columns, followed by affinity-based purification. In brief, plasma was incubated with anti-HBsAg, and qEV columns were used to remove unbound anti-HBsAg, serum impurities and the majority of HBV virions. Together, with the use of affinity-based purification, this protocol led to the complete depletion of anti-HBsAg bound particles (e.g. virions and filaments) from EV preparations.
Key findings: The combination of qEV columns with affinity-based purification ensured purity of EV samples free from virus-sized particles, hepatitis B surface antigen and core antigen, antibody contaminations, and infectious potential.
Exploring biomarkers for hepatocellular carcinoma: distinguishing free protein from exosomal biomarkers
Paper: Easom N, Marks M, Jobe D, et al. (2020). ULBP1 is elevated in human hepatocellular carcinoma and predicts outcome. Frontiers in Oncology. 10:971. doi: 10.3389/fonc.2020.00971
Background: Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide. The most common underlying cause is hepatitis B, followed by hepatitis C and other liver diseases. This study examined a ligand of a receptor involved in potentially druggable cancer pathways (Natural Killer cell Group 2 receptor D, NKG2D).
Study approach: This study was focused on a binding protein (and NKG2D ligand) called ULBP1, which is expressed by cells undergoing viral infection. To understand the nature of circulating ULBP1, Easom et al. (2020) compared the concentration of ULBP1 (a NKG2D ligand) in exosomes and as free protein, in the circulation of patients with HCC. This was achieved by comparing serum separated by Izon’s qEV columns.
Key findings: ULBP1 concentration was elevated in the serum of patients with HCC, compared with either patients with cirrhosis or with healthy controls – highlighting the diagnostic potential of ULBP1. Rather than being bound to exosomes, ULBP1 was present predominantly as free protein.
Investigating miRNA barcodes in extracellular vesicles as markers of alcohol-related liver injury
Paper: Eguchi A, Franz N, Kobayashi Y et al. (2019). Circulating extracellular vesicles and their miR “barcode” differentiate alcohol drinkers with liver injury and those without liver injury in severe trauma patients. Frontiers in Medicine 6: Article 30
Background: Although not an official focus of World Hepatitis Day, alcohol hepatitis is a major health concern. One of the many implications of severe liver injury is that it is associated with pro-inflammatory reactions – a particular concern when treating patients in the emergency department. As prompt recognition of historical alcohol abuse can be lifesaving in the clinic, the development of non-invasive tools for assessing liver injury in intoxicated trauma patients is of interest. To this end, Eguchi et al. (2019) embarked on a study to examine whether EV-microRNAs reflect liver injury in alcohol-intoxicated trauma patients.
Study approach: Eguchi et al. (2019) compared expression levels of a panel of microRNAs in circulating EVs, isolated with qEV columns, across different groups: non-drinkers, alcohol drinkers with liver injury, and alcohol drinkers without liver injury. The microRNA panel was compared and analysed alongside patient outcomes.
Key findings: EV concentration and expression levels of specific EV-miRNAs are released by damaged hepatocytes and correlate with serum bilirubin, providing support for the hypothesis that EVs could be used to identify patient drinking behaviour in trauma patients.
Purification technology enabling advances in virology research
The isolation of EV or virus particles to a high level of purity and concentration is critical to their study. As highlighted here, and elsewhere<super-script>4<super-script>, independent groups have shown that Izon’s qEV columns remove contaminants to a greater extent than other methods, such as ultracentrifugation. Efficient, qEV-based separation of EVs and virus particles plays a significant role in advancing important EV and virology research, such as the hepatitis-related studies highlighted here.
To learn about important considerations for the purification and quantification of viruses and virus-like particles, download this app note now: Viruses and VLPs in Nanomedicine: Importance of Separation and Quantification
