Van Latentie tot Ziekte: Epstein-Barr-Virus in Transplantatiepatiënten

Epstein-Barr virus (EBV) is a double-stranded DNA virus belonging to the γ-Herpesvirinae subfamily. It is a highly prevalent virus, infecting over 95% of the word population. EBV primary infection typically occurs during early childhood and is usually asymptomatic, whereas infection in young adults may result in infectious mononucleosis (IM). EBV is mainly transmitted through saliva, initially infecting epithelial cells before establishing latency in B-cells. In the latent state, EBV persists life-long with minimal antigen expression, enabling the evasion of immune system surveillance. Under immunosuppressive conditions, EBV can reactivate and contribute to the development of life-threatening malignancies such as Burkitt's Lymphoma (BL), Hodgkin Lymphoma (HL), and post-transplant lymphoproliferative disorder (PTLD). EBV infections and associated disorders are responsible for approximately 200,000 new cancer cases each year. Moreover, EBV and other human herpesviruses (HHVs), such as cytomegalovirus (CMV) and herpes simplex virus (HSV-1), infections are common in post-transplant patients and remain an important cause of morbidity and mortality among solid organ transplant (SOT) and hematopoietic stem cell transplant (HSCT) recipients, and can cause serious complications. One of these complications, is PTLD, a major life-threatening post-transplant disorder developing due to a compromised T-cell surveillance allowing EBV-infected B-cells to proliferate uncontrollably. In this thesis, we aimed to advance the current understanding of HHV infections, with focus on EBV, in transplant recipients. In Chapter 3 and 4, we investigated novel, non-invasive potential biomarkers for both PTLD diagnosis and disease monitoring, whereas in Chapter 5 and 6 we wished to gain a deeper understanding of EBV infection and other HHVs and/or adenovirus (AdV) multiple infections in transplant recipients by investigating their co-infections, the emergence of drug-resistance and of novel mutations of undetermined significance. In Chapter 3, we investigated whether other viruses, in addition to EBV, could provide additional diagnostic or prognostic value in PTLD. Specifically, we analyzed the kinetics of post-transplant viral infections, including Torque Teno Virus (TTV), EBV, CMV, human herpes virus 6 (HHV-6) and AdV, in HSCT recipients with or without PTLD at 3 different timepoint. TTV, a DNA virus belonging to the Anelloviridae family, has recently gained interest as a potential biomarker of immunosuppression since high TTV viral load has been associated with increasing immunosuppression, whereas low TTV levels are linked to immune reconstitution. Thus, by monitoring TTV viral load we could potentially assess the patient's immune status. Our findings demonstrated significantly higher TTV levels in PTLD patients compared to healthy donors and post-HSCT patients who did not develop PTLD with a continuous rise in TTV levels seen in PTLD-HSCT throughout the timepoints, indicating that TTV levels increase with the fall of the host immunocompetence. Therefore, our results point to the usefulness of TTV viral loads in post-HSCT recipients as a marker of immune competence and its potential use to tailor immune suppression in the post-transplant setting. In Chapter 4, we analyzed the expression of 28 miRNAs (13 EBV-encoded and 15 host-encoded miRNAs) in exosomes derived from PTLD patients following SOT or HSCT. The mechanisms of EBV immune evasion, are still not completely understood. Modulation of the expression of host microRNAs (miRNAs), which are small non-coding RNAs that regulate various biological processes, can help to avoid immune recognition. EBV-encoded miRNAs support the survival of tumor cells and can manipulate the expression of host miRNAs to evade immune detection. These miRNAs can be packaged, by EBV-infected cells, into exosomes, which are small extracellular vesicles released by cells into body fluids, and can be transferred throughout the body, making them promising candidates for non-invasive biomarkers. While no significant differences were observed between post-HSCT PTLD and Non-PTLD patients, distinct miRNA expression profile was identified when comparing PTLD patients, both post-SOT and post-HSCT, to healthy donors. Additionally, we detected significant differences in miRNA expression between EBV positive and EBV negative PTLD patients post-SOT. Future research should focus on detailed characterization of additional components of the exosomal cargo to identify reliable and non-invasive biomarkers that are critical and specific for PTLD development. Chapter 5 and 6 focused on investigating HHVs multiple (co-)infections and antiviral resistance in immunocompromised patients to gain better understanding of their potential interactions. Multiple HHVs (co-)infections are associated with more clinical complications and higher mortality in post-transplant patients, despite antiviral treatment. Moreover, there is still a major problem for immunocompromised patients developing antiviral drug-resistance, which further complicated patient management. EBV frequently reactivates in immunosuppressive conditions, however, data on EBV genetic variability during antiviral therapy and on the impact on other DNA virus infections remains limited. In Chapter 5, we conducted a longitudinal analysis of a HSCT recipient with simultaneous EBV and CMV reactivations by analyzing 22 samples during 8 months while under antiviral treatment. Viral dynamics of CMV, EBV and TTV and the emergence of mutations were monitored by genotyping via Sanger sequencing (prospectively) and next-generation sequencing (NGS) (retrospectively). EBV reactivation was seen simultaneously with CMV reactivation and rising of TTV DNA load, suggesting increased immunosuppression. NGS enabled earlier detection of CMV-drug-resistance mutations compare to Sanger sequencing, underscoring its clinical utility. Moreover, a reduction in EBV viremia was observed during maribavir therapy, suggesting that this antiviral may be of benefit in transplant recipients with active EBV replication. Our findings highlight the difficulties in managing CMV/EBV co-reactivation in immunocompromised hosts. In Chapter 6, we expanded the analysis of HHVs multiple (co-)infections to a cohort of 84 immunocompromised patients. We assessed the prevalence of EBV in patients having consecutively or concomitant multiple HHV and/or AdV infections as well as emergence of drug resistance. Prospective genotyping by Sanger sequencing and/or phenotyping allowed the identification of HHVs and AdV drug-resistant mutations. Retrospective analysis was conducted to assess EBV prevalence and identify EBV genetic variability. CMV was the most prevalent infection, followed by HSV-1 in patients having multiple infections during their follow-up. Although EBV prospective testing was performed only in 6 patients, retrospective analysis identified EBV in 26 additional patients. The highest incidence of drug resistance was found for HSV-1. Various novel mutations of undetermined significance were also identified in different HHVs and AdV. These findings highlight the under-recognition of EBV infection in clinical practice despite its oncogenic potential and the fact that HHV infections in immunocompromised patients are still a clinical challenge, with emergence of antiviral resistance and the occurrence of multiple infections further complicating treatment. In conclusion, this thesis aimed to demonstrate that EBV, and other HHVs, infection remain a significant challenge in the management of transplant recipients, due to reactivation, co-infections and the emergence of antiviral resistance. Furthermore, this work highlights the unmet need for reliable, non-invasive EBV-associated biomarkers for PTLD diagnosis, prognosis, or response to treatment, which creates a pitfall for early disease diagnosis and monitoring. Overall, EBV infection in immunocompromised patients remains a significant clinical and research challenge due to the complex interplay of EBV with the host immune system, other HHVs infections, and the ability to drive the development of life-threatening complications. Therefore, further research is required for better understanding EBV biology and advance the development of strategies to prevent and to control EBV infections in the immunocompromised host.