Extracellular vesicle phenotypic patterns associated with CMV, EBV and HHV-6 reactivation after haploidentical transplantation with post-transplant cyclophosphamide.

Introduction: Human cytomegalovirus (CMV), Epstein-Barr virus (EBV), and human herpesvirus 6 (HHV-6) remain important causes of morbidity after allogeneic hematopoietic cell transplantation (HCT), yet biomarkers predicting viral reactivation are lacking. Extracellular vesicles (EVs) have emerged as promising liquid-biopsy tools in inflammatory and infectious diseases. Methods: In this exploratory biomarker discovery study, 32 consecutive recipients of haploidentical HCT with post-transplant cyclophosphamide were prospectively enrolled. EV surface marker profiles were assessed by flow cytometry before transplantation and at predefined time points during the first three months post-HCT. Associations between EV phenotypes and viral reactivation were evaluated using logistic regression and time-varying Cox models. Results: CMV, EBV, and HHV-6 reactivation occurred in 20, 11, and 17 patients, respectively. Multivariable analyses showed that different EV phenotypic patterns were associated with CMV, EBV, and HHV-6 reactivation. CMV reactivation was associated with increased EV expression of CD44, CD86, CD140a, and CD144. EBV reactivation correlated with reduced CD30-positive EV concentrations and increased KRT18 expression. HHV-6 reactivation was associated with altered CD26, CD86, CD120a, and CD144 EV profiles. These associations remained significant after adjustment for acute graft-versus-host disease and concomitant viral infections. Discussion: Collectively, these exploratory findings support further validation of circulating EV phenotypes as candidate biomarkers for predicting and monitoring viral reactivation after haploidentical HCT.