HHV6 Specific T-Cells Are Predictive Biomarker of Active HHV6 Infection after Allogeneic Hematopoietic Stem Cell Transplantation: Results of a Prospective Study in 213 Patients
Authors: Greco Raffaella , Noviello Maddalena , Crucitti Lara , Racca Sara , Valtolina Veronica , Morelli Mara , Giglio Fabio , Mannina Daniele , Barbanti Maria Chiara , Forcina Alessandra , Pozzato Mattia , Rolla Serena , Assanelli Andrea Angelo , Carrabba Matteo Giovanni , Marktel Sarah , Bernardi Massimo , Corti Consuelo , Lupo Stanghellini Maria Teresa , Vago Luca , Peccatori Jacopo , Clementi Massimo , Ciceri Fabio , Bonini Chiara 
Published: October 2019

Abstract
BACKGROUND: Although Human herpesvirus 6 (HHV6) reactivation in healthy individuals usually occurs without significant morbidity, in recipients of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is associated with severe clinical manifestations and increased transplant-related mortality (TRM). The role of HHV6 in transplant-related complications remains in question, considering that both latent and active viral infection can occur. Moreover, only limited experiences are reported on HHV6-specific immune responses after HSCT, and their correlation with clinical outcome is largely unexplored.
METHODS: From February 2013 to October 2015, we conducted a prospective observational study to investigate HHV6 reactivation in 213 consecutive adult patients (median age 52 years) who received allo-HSCT for high-risk hematological malignancies (57% acute leukemia) in our institute. Stem cell donors were family haploidentical (104), HLA identical sibling (39), unrelated (63), cord blood (7). Stem cell source was mainly T-cell replete PBSCs (87%). Viral load was weekly monitored by quantitative PCR in plasma within the first month after HSCT. Numbers of IFNγ-producing HHV6-T-cells were determined by enzyme-linked immunospot assay (ELISPOT). We challenged patients PBMC against a library of overlapping peptides covering the entire sequence of the immunodominant virus protein U54, expressed during the lytic cycle of virus replication. Patients were evaluated at a median of 34 days after HSCT (HHV6-; 57 patients) for controls or by the 4th day after the first HHV6 DNAemia (median 32 days) for reactivating patients (HHV-6+; 54 patients).
RESULTS: HHV6-reactivation occurred in 56% of patients at 100 days, with a median time of 28 days after HSCT. HHV6 was detected in plasma for 86% of patients, while 33% resulted positive in other materials: 9 BM aspirates, 39 gut biopsies, 3 BAL, 5 CSF. All patients received acyclovir as prophylaxis. Only 41% of reactivating patients presented a clinically relevant HHV6 infection (HHV6 positivity in presence of HHV6-related clinical symptoms and/or HHV6-disease). Clinical manifestations were: fever (25), skin rash (37), hepatitis (19), diarrhoea (28), encephalitis (5), BM suppression (30). According to center guidelines, antiviral treatment was given in 23% of reactivating patients, for uncontrolled clinically relevant HHV6 infection.
Overall survival (OS) was not different in HHV6 reactivating patients compared to controls (p=0,2). Relapse incidence and TRM were not affected by HHV6. All HSCT recipients showed a better OS with CD3+ cells≥200/mcl at 30 days (p <0.001), independently of HHV6. In univariate analysis, we identified the following risk factors for HHV6-reactivation: active disease status before HSCT (p=0,052), haploidentical HSCT (p=0,003), PT-Cy use (p <0.001), CMV reactivation (p=0,001), GvHD (p=0,003), CD3+ cells<200/mcl at 30 days (p=0,013).
The number of IFNγ-producing HHV6-specific T-cells was significantly higher in HHV6 reactivating patients (p= 0.0149; mean number of specific T-cells 43.48 per 10^5 PBMC) than in non-reactivating patients (specific T-cells 12.57 per 10^5 PBMC), especially in the presence of active and clinically relevant HHV6 infection (p<0,0001; mean number of specific T-cells 81.46 per 10^5 PBMC). No influence of IFNγ-producing CMV specific T-cells, absolute counts of CD3+ T cells or GvHD was observed.
CONCLUSIONS: In this study, we observed that active disease status before HSCT, haploidentical donors, especially using PT-Cy, CMV reactivation, GvHD and lower CD3+ counts at 30 days, are strong predictors of HHV6 reactivation. HHV6-specific T-cells, detectable by ELISPOT assay despite extremely low T-cell numbers and immunosuppressive therapy, are significantly associated with active and clinically relevant HHV6 infections, representing a new and promising tool to unravel the role of HHV6 positivity in allo-HSCT recipients.

Disclosures
Ciceri: MolMed SpA: Consultancy. Bonini:TxCell: Membership on an entity's Board of Directors or advisory committees; Molmed SpA: Consultancy.