Adoptive T-Cell Therapy to Prevent and Treat Human Metapneumovirus (hMPV) Infections Post Hematopoietic Stem Cell Transplant (HSCT)

Viral infections are a significant cause of morbidity and mortality in allogeneic HSCT recipients. Previous studies by our group and others have demonstrated that CMV, EBV, AdV, BK and HHV6 infections can be treated using adoptively transferred in vitro expanded virus-specific T-cells (VSTs). However, the list of pathogens linked with high morbidity/mortality continues to grow, while VST therapy has been limited by the paucity of information regarding immunogenic/protective T-cell target antigens within many viruses. The goal of this study was to characterize the cellular immune response to human metapneumovirus (hMPV), a paramyxoviridae detected in up to 10% of HSCT recipients and associated with mortality of up to 43%.

To identify immunodominant T-cell targets we assessed the cellular immune response directed against all 9 antigens expressed by hMPV (N, M, F, SH, G, M2-1, M2-2, P, L). For T-cell stimulation we isolated PBMCs from healthy donors, pulsed them with overlapping peptide libraries (15mers overlapping by 11aa) spanning each antigen, and then expanded them in vitro for 10 days. The resulting lines were polyclonal with a predominance of CD4+ T-cells (mean 61+/-3%, n=24). To assess the specificity we used an IFNg ELIspot assay and defined a hierarchy of immunodominance according to the number of responders and the magnitude of response. F was most frequently recognized (n=26) and induced the highest frequency of specific cells (mean 369±53 SFC/2x10⁵), followed, in descending order, by N (n=23; mean 238±26), M (n=23; mean 169±36), M2-1 (n=22; mean 153±27), P (n=20; mean 167±34), G (n=10; mean 60±15) and L (n=8; mean 107±19). Reactivity against SH and M2-2 was detected in 3 and 1 donors, respectively. The VSTs were polyfunctional, producing multiple cytokines (IFNγ, TNFα, GM-CSF) and effector molecules (Granzyme B) upon stimulation with cognate antigen, as evaluated by intracellular cytokine staining, luminex and ELIspot. Finally, we were able to detect T-cells specific for F, N, M2-1 and P in the peripheral blood of a patient who cleared their hMPV infection after HSCT, proving the in vivo relevance of our findings. In summary we were able to generate hMPV-VSTs and characterize the T-cell immune response directed against the encoded viral proteins in healthy seropositive donors. We next plan to assess the cytolytic capacity of our hMPV-VSTs against virus-infected targets, identify immunodominant epitopes and ultimately generate clinical hMPV-VSTs for adoptive transfer in patients after allogeneic HSCT.