Identification of clinically relevant CD4+ T-cell epitopes from HCMV antigens

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Dokumentart: PhDThesis
Date: 2023-01-12
Language: English
Faculty: 4 Medizinische Fakultät
Department: Medizin
Advisor: Stevanovic, Stefan (Prof. Dr.)
Day of Oral Examination: 2022-11-29
DDC Classifikation: 610 - Medicine and health
Keywords: Cytomegalie-Virus , Epitop , T-Lymphozyt , HLA , Peptide , Immuntherapie
Other Keywords:
promiscuous CD4+ T-cell epitopes, epitope prediction, antiviral T cells, HCMV, Human Cytomegalovirus, peptide pools, adoptive T-Cell therapy, HLA class II
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Sixty percent of our population encountered Human Cytomegalovirus (HCMV), followed by a life-long persistence of the virus in the host. Immunotherapies, like the adoptive transfer of HCMV-specific T cells, have been shown to cure HCMV disease in patients susceptible to viral infection or reactivation due to immunosuppression. The adoptive T-cell transfer depends on comprehensive knowledge about target antigens and T-cell epitopes presented on HLA molecules. HCMV-specific CD4+ T cells efficiently control viral infection. However, CD4+ T-cell epitopes have only been identified for a few immunodominant antigens of HCMV so far. In this project, we screened the entire proteome of HCMV for frequently recognized promiscuous CD4+ T-cell epitopes using in silico epitope prediction. We tested 103 predicted epitope candidates for immunogenicity in PBMCs of HCMV-seropositive donors in ELISpot assays after a 12-day stimulation with peptide and IL-2. This way, we identified 74 T-cell epitopes from 58 different source proteins. The spectrum of source proteins covers all temporal stages of gene expression and comprises various functional profiles. Seventeen of the 74 epitopes were immunodominant (recognized by > 50% of the tested PBMC samples) and have not been published before. We validated the dominant T-cell epitopes by intracellular cytokine staining, i.e., we confirmed IFNγ and TNF release of CD4+ T cells after stimulation with the epitopes. Subsequently, we generated a peptide pool containing 14 dominant epitopes, including three previously published epitopes. The peptide pool triggered T-cell activation in 46 of 48 randomly selected PBMC samples. Another peptide pool, lacking the pp65-derived peptides, achieved an analogous recognition rate of 30 out of 30. Our pool of promiscuous dominant epitopes stimulates HCMV-specific T cells of blood donors with different HLA polymorphisms, circumventing the elaborate design of individualized therapy. The novel T-cell epitopes can be directly translated into the clinic to improve T-cell therapy.

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