Background: Adoptive transfer of genetically engineered T cells expressing antigen-specific T-cell receptors (TCRs) is an appealing therapeutic approach for Epstein-Barr virus (EBV)-associated... Show moreBackground: Adoptive transfer of genetically engineered T cells expressing antigen-specific T-cell receptors (TCRs) is an appealing therapeutic approach for Epstein-Barr virus (EBV)-associated malignancies of latency type II/III that express EBV antigens (LMP1/2). Patients who are HLA-A*01:01 positive could benefit from such products, since no T cells recognizing any EBV-derived peptide in this common HLA allele have been found thus far. Methods: HLA-A*01:01-restricted EBV-LMP2-specific T cells were isolated using peptide major histocompatibility complex (pMHC) tetramers. Functionality was assessed by production of interferon gamma (IFN-gamma) and cytotoxicity when stimulated with EBV-LMP2-expressing cell lines. Functionality of primary T cells transduced with HLA-A*01:01-restricted EBV-LMP2-specific TCRs was optimized by knocking out the endogenous TCRs of primary T cells ( increment TCR) using CRISPR-Cas9 technology. Results: EBV-LMP2-specific T cells were successfully isolated and their TCRs were characterized. TCR gene transfer in primary T cells resulted in specific pMHC tetramer binding and reactivity against EBV-LMP2-expressing cell lines. The mean fluorescence intensity of pMHC-tetramer binding was increased 1.5-2 fold when the endogenous TCRs of CD8(+) T cells was knocked out. CD8(+/ increment TCR) T cells modified to express EBV-LMP2-specific TCRs showed IFN-gamma secretion and cytotoxicity toward EBV-LMP2-expressing malignant cell lines. Conclusions: We isolated the first functional HLA-A*01:01-restricted EBV-LMP2-specific T-cell populations and TCRs, which can potentially be used in future TCR gene therapy to treat EBV-associated latency type II/III malignancies.Here we identify the first HLA-A*01:01-restricted Epstein-Barr virus Latent Membrane Protein 2 (EBV-LMP2)-specific T-cell population and show that these T-cell populations and T cells modified to express the LMP2-specific T-cell receptor showed IFN-gamma secretion and cytotoxicity toward EBV-LMP2-expressing malignant cell lines. Show less
The human herpesvirus Epstein-Barr virus (EBV) is a large DNA virus that infects over 90% of the adult world population. EBV is the causative agent of infectious mononucleosis and EBV infection is... Show moreThe human herpesvirus Epstein-Barr virus (EBV) is a large DNA virus that infects over 90% of the adult world population. EBV is the causative agent of infectious mononucleosis and EBV infection is associated with various malignancies. EBV establishes lifelong infections in immunocompetent hosts. To counteract the host’s immune defence, EBV acquired numerous immune evasion mechanisms. During latency of EBV, viral protein synthesis is limited or absent, making the virus-infected cells virtually invisible to the immune system. Evasion mechanisms of EBV active during primary infection as well as in reactivation are necessary for establishment of latent infection and prolonged replication. Studying viral evasion not only helps to understand EBV, but also the human immune system. Viral molecules interfering with antigen presentation by HLA I and HLA II have been identified previously, but so far, it was unclear how EBV interferes with the lipid antigen-presenting molecule CD1d. The work described in this thesis shows EBV’s mechanism to interfere with cell surface expression of CD1d. Further, a novel immune evasion molecule that obstructs antigen-presentation during the late lytic phase of EBV infection was identified and its working mechanism was unravelled. Understanding viral immune evasion mechanisms may aid in developing therapies for EBV-associated diseases. Show less
Real-time monitoring of PCR has strongly supported the increased diagnostic use of nucleic acid detection assays in clinical virology. Particularly the improvements in the ability to quantify... Show moreReal-time monitoring of PCR has strongly supported the increased diagnostic use of nucleic acid detection assays in clinical virology. Particularly the improvements in the ability to quantify target nucleic acid sequences offer new opportunities in the management of viral infections. Real-time PCR is rapidly replacing traditional PCR, and new diagnostic uses will likely emerge. This thesis explores the wide range of potential applications of real-time quantitative PCR technology in clinical virology. This exploration is directed to the design of methods, the application to relevant patient categories, the comparison with established methods where available, and the definition of the clinical relevance of the approach. The focus comprises viral targets where an elaborate balance between viral replication and the host immune system has been established, which brings about viral maintenance without affecting the host, until this balance is disturbed. Show less
