Caratterizzazione di genomi virali mediante spettrometria di massa MALDI-TOF in nanovescicole sieriche e urinarie isolate da pazienti sottoposti a trapianto renale

Advances in surgical techniques and the evolution of immunosuppressive therapy have undoubtedly improved survival rates, but post-transplant infections continue to cause morbidity and mortality in these patients. Exosomes, nanometric vesicles released by most of the cells present in the various biological fluids, can transfer their load of proteins and nucleic acids to the receiving cells. The aim of this study was to evaluate the presence of viral genomes, belonging to the families of PyVs and HPVs, associated with nanovescicles, in 72 patients undergoing renal transplantation, in samples of blood and urine. The identification of viral genomes was carried out through the development of a high-throughput method based on mass spectrometry MALDI- TOF. The method allowed the simultaneous detection of 18 viral genomes for PyV and 16 HPV for each sample tested. The isolation of the NV was initially carried out using a precipitation method based on PEG. The number and size of the isolated NV was determined by NTAs. The determination of exosomal epitopes was performed by the MACSplex method. The enrichment of the exosomial fraction in the urinary NVs was revealed by the high presence of the CD9 antigen on the surface of the NVs themselves. Moreover, the use of enzymatic digestion allowed to test how unspecific the detection of viral genomes identified with mass spectrometry could be, in case of unexpected copurifications. The results obtained on the transplanted patients have shown marked positive percentages for the viral genome of BKV (60%) and JCV (20%) in serum NV but especially in urinary ones (50% both). In conclusion, the investigation carried out in this project has led to the identification of the combination of isolation by immunomagnetic separation and enzyme treatment with DNAase I as the best method for the determination of viral genomes contained within exosomes (.i.e. SV12).