Abstract
Morbidity and mortality of immunocompromised patients are increased by primary infection with or reactivation of Epstein-Barr virus (EBV), possibly triggering EBV+ post-transplant lymphoproliferative disease (PTLD). Adoptive transfer of EBV-specific cytotoxic T cells (EBV-CTLs) promises a non-toxic immunotherapy to effectively prevent or treat these complications.To improve immunotherapy and immunomonitoring this study aimed at identifying and evaluating naturally processed and presented HLA-A*03:01-restricted EBV-CTL epitopes as immunodominant targets. More than 15000 peptides were sequenced from EBV-immortalized B cells transduced with soluble HLA-A*03:01, sorted using different epitope prediction tools and eleven candidates were preselected. T2 and Flex-T peptide-binding and dissociation assays confirmed the stability of peptide-MHC complexes. Their immunogenicity and clinical relevance were evaluated by assessing the frequencies and functionality of EBV-CTLs in healthy donors (n > 10) and EBV+ PTLD-patients (n = 5) by multimer staining, Eli- and FluoroSpot assays. All eleven peptides elicited EBV-CTL responses in the donors. Their clinical applicability was determined by small-scale T-cell enrichment using Cytokine Secretion Assay and immunophenotyping. Mixtures of these peptides when added to the EBV Consensus pool revealed enhanced stimulation and enrichment efficacy. These EBV-specific epitopes broadening the repertoire of known targets will improve manufacturing of clinically applicable EBV-CTLs and monitoring of EBV-specific T-cell responses in patients.
Highlights
After hematopoietic stem cell (HSCT) and solid organ transplantation (SOT), patients are rendered highly susceptible to viral infections and reactivations due to transplant-related immunosuppressive therapy and delayed immune reconstitution
A combination of different epitope prediction tools was applied to scan the unfiltered sequences of HLAA*03:01-restricted Epstein-Barr virus (EBV)-derived peptides isolated in vivo
A total of about 15,000 unfiltered EBV-specific peptide-sequences were isolated in vivo (Supplementary Figure 1)
Summary
After hematopoietic stem cell (HSCT) and solid organ transplantation (SOT), patients are rendered highly susceptible to viral infections and reactivations due to transplant-related immunosuppressive therapy and delayed immune reconstitution. Viral replication cannot be controlled due to insufficient EBV-specific immunity. Reactivation of EBV in HSCT recipients ranges from 10 to 50%, while the incidences in SOT recipients vary from 1 to 20% depending on risk factors like EBV-serostatus, age at transplantation, immunosuppressive regimen post-transplant and organ graft [3, 5, 6]. EBV-seronegative patients with EBV-seropositive donors [7, 9] and EBV-seronegative transplant recipients suffering from primary EBV-infection in a post-transplant immunosuppressed setting [10, 11] carry the highest risk for PTLD development. It is likely to remain ineffective due to several host- and tumor-related mechanisms [5, 19,20,21,22] and frequently leads to PTLD recurrences [5, 23]
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