Discovery of immunodominant T-cell epitopes reveals penton protein as a second immunodominant target in human adenovirus infection

Additional file 2: Figure S2. Analysis of HAdV-specific T-cell responses against the novel immunodominant T-cell epitope in healthy donors

Diagnostic Parameters of Adenoviremia in Pediatric Stem Cell Transplant Recipients

Despite recent progress in the diagnostic risk assessment of human adenovirus (HAdV) infections in immunocompromised patients, clinical complications mediated by these viruses continue contributing to significant morbidity and mortality, particularly in the pediatric hematopoietic allogeneic stem cell transplant (HSCT) setting. Current data highlight the importance of monitoring stool samples to assess the risk of invasive HAdV infections in children undergoing HSCT. The advent of novel, more effective antiviral treatment options might permit successful virus control even at the stage of systemic infection, thus increasing the interest in optimized HAdV monitoring in peripheral blood (PB). We have screened over 300 pediatric HCST recipients by serial monitoring of stool and PB specimens, and identified 31 cases of invasive HAdV infection by quantitative pan-adenovirus RQ-PCR analysis of consecutive PB specimens. The diagnostic parameters assessed included HAdV peak levels (PL) and the time-averaged area under the curve (AAUC) of virus copy numbers. The predictive value for patient outcome reflected by non-relapse and HAdV-related mortality was determined. The patients were assigned to quartiles based on their PL and AAUC, and the readouts were highly correlated (p < 0.0001). Non-relapse mortality in patients by AAUC quartile (lowest to highest) was 26, 50, 75, and 86%, respectively, and AAUC was strongly correlated with non-relapse mortality (p < 0.0001), while the association between PL and non-relapse mortality was less pronounced (p = 0.013). HAdV-related mortality was absent or very low in patients within the two lower quartiles of both PL and AAUC, and increased to ≥70% in the upper two quartiles. Despite the significant correlation of PL and AAUC with patient outcome, it is necessary to consider that the risk of non-relapse mortality even within the lowest quartile was still relatively high, and it might be difficult therefore to translate the results into differential treatment approaches. By contrast, the correlation with HAdV-related mortality might permit the identification of a low-risk patient subset. Nevertheless, the well-established correlation of HAdV shedding into the stool and intestinal expansion of the virus with the risk of invasive infection will expectedly remain an essential diagnostic parameter in the pediatric HSCT setting

Frontiers in Microbiology / Post-transplant Replication of Torque Teno Virus in Granulocytes

Torque Teno virus (TTV) in humans is characterized by ubiquitous occurrence in peripheral blood (PB), without any related disease described to date. Several studies reported a significant increase of TTV plasma DNA levels in allogeneic transplant recipients, and suggested a correlation of elevated virus titers with immunosuppression and transplant-related complications. However, the site of viral replication in this setting has remained unclear. We have studied TTV in serial plasma specimens derived from 43 pediatric allogeneic hematopoietic stem cell transplantation (HSCT) recipients by RQ-PCR, and found increasing TTV-DNA levels in all patients post-transplant, with a peak around day +100 and maximum virus copy numbers reaching 4 10E9/ml. To assess whether the virus replicates in PB-cells, leukocyte subsets including granulocytes, monocytes, NK-cells, T- and B-lymphocytes were serially isolated by flow-sorting for TTV analysis in 19 patients. The virus was undetectable in most cell types, but was identified in granulocytes in all instances, revealing a median DNA copy number increase of 1.8 logs between days +30100 post-transplant. Our data therefore provide evidence for TTV replication in granulocytes in this setting. In a control cohort of immunocompetent children and in HSCT recipients before day +30, TTV positivity in granulocytes was less common (33%), and the copy numbers were considerably lower. However, rising TTV replication about 2 weeks after granulocyte engraftment (>500 cells/l) was observed suggesting that granulocyte recovery might be required for TTV expansion in severely immunosuppressed transplant recipients.(VLID)470320

Short-Term In-Vitro Expansion Improves Monitoring and Allows Affordable Generation of Virus-Specific T-Cells against Several Viruses for a Broad Clinical Application

<div><p>Adenoviral infections are a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT) in pediatric patients. Adoptive transfer of donor-derived human adenovirus (HAdV)-specific T-cells represents a promising treatment option. However, the difficulty in identifying and selecting rare HAdV-specific T-cells, and the short time span between patients at high risk for invasive infection and viremia are major limitations. We therefore developed an IL-15-driven 6 to 12 day short-term protocol for <i>in vitro</i> detection of HAdV-specific T cells, as revealed by known MHC class I multimers and a newly identified adenoviral CD8 T-cell epitope derived from the E1A protein for the frequent HLA-type A*02∶01 and IFN-γ. Using this novel and improved diagnostic approach we observed a correlation between adenoviral load and reconstitution of CD8⁺ and CD4⁺ HAdV-specific T-cells including central memory cells in HSCT-patients. Adaption of the 12-day protocol to good manufacturing practice conditions resulted in a 2.6-log (mean) expansion of HAdV-specific T-cells displaying high cytolytic activity (4-fold) compared to controls and low or absent alloreactivity. Similar protocols successfully identified and rapidly expanded CMV-, EBV-, and BKV-specific T-cells. Our approach provides a powerful clinical-grade convertible tool for rapid and cost-effective detection and enrichment of multiple virus-specific T-cells that may facilitate broad clinical application.</p></div

Alloreactive potential of PBMCs versus expanded control- and seHAdV-specific T-cells.

<p>CFSE-labeled autologous or allogeneic responder cells (PBMCs or seHAdV-T-cells) were mixed 1∶1 with irradiated stimulator cells (PBMCs) and incubated for 7 to 8 days. A) The graph shows the total number of proliferated viable cells per 96 well plate of 14 different donor/recipient combinations A–N). Proliferation of seHAdV-T-cells or PBMCs in response to autologous (auto) or allogeneic (allo) irradiated PBMCs are shown. In addition, representative examples from donors A and J are given. B) A summarizing graph show mean + SEM from all combinations (except for L,M and N); The percentage of viable proliferating seHAdV-T-cells was set to 100% and calculated based on total cell number. C) seHAdV-T-cells or seMAGE-1A-T-cells (control) and PBMC were mixed with allogeneic irradiated stimulator PBMCs. A summarizing graph shows mean + SEM of 4 combinations. First, total cell number of viable proliferating (CFSE-low) seHAdV-, seMAGE-1A-T-cells and PBMCs/well are calculated. Based on these total cell number, the percentage values were analyzed and compared to allogeneic PBMCs, which was set to 100%. A representative histogram of one donor is shown, including percentage values of proliferated cells. Significance vs HAdV-T-cell line: o, p< = 0.07; *, p< = 0.01.***, p< = 0.001.</p

Phenotypic analysis of seHAdV-T-cells.

<p>A) Percentage values of different cell populations (as indicated, including cytokine-induced killer cells (CIK)) within PBMCs, before (day 0) and after expansion (day12) of 8 donors. Percentage values of TCM (upper left), Naïve (upper right), TEM (lower left) and TEMRA (lower right) T cell populations within CD8⁺ (B), CD4⁺ C), and HAdV-streptamer⁺ T-cells D) on days 0 (white bars) and 12 (black bars). The graph shows mean+SEM of 8 (for CD4⁺ and CD8⁺ T-cells) and 3 (for HAdV-streptamer⁺ T-cells) donors. Notably, for phenotypic analysis of HAdV-streptamer⁺ T cells on day 0 (white bars), beads-based magnetic isolation was performed. Representative dot plots are shown.</p