Therapeutic Vaccination of Hematopoietic Cell Transplantation Recipients Improves Protective CD8 T-Cell Immunotherapy of Cytomegalovirus Infection

Abstract

Reactivation of latent cytomegalovirus (CMV) endangers the therapeutic success of hematopoietic cell transplantation (HCT) in tumor patients due to cytopathogenic virus spread that leads to organ manifestations of CMV disease, to interstitial pneumonia in particular. In cases of virus variants that are refractory to standard antiviral pharmacotherapy, immunotherapy by adoptive cell transfer (ACT) of virus-specific CD8+ T cells is the last resort to bridge the “protection gap” between hematoablative conditioning for HCT and endogenous reconstitution of antiviral immunity. We have used the well-established mouse model of CD8+ T-cell immunotherapy by ACT in a setting of experimental HCT and murine CMV (mCMV) infection to pursue the concept of improving the efficacy of ACT by therapeutic vaccination (TherVac) post-HCT. TherVac aims at restimulation and expansion of limited numbers of transferred antiviral CD8+ T cells within the recipient. Syngeneic HCT was performed with C57BL/6 mice as donors and recipients. Recipients were infected with recombinant mCMV (mCMV-SIINFEKL) that expresses antigenic peptide SIINFEKL presented to CD8+ T cells by the MHC class-I molecule Kb. ACT was performed with transgenic OT-I CD8+ T cells expressing a T-cell receptor specific for SIINFEKL-Kb. Recombinant human CMV dense bodies (DB-SIINFEKL), engineered to contain SIINFEKL within tegument protein pUL83/pp65, served for vaccination. DBs were chosen as they represent non-infectious, enveloped, and thus fusion-competent subviral particles capable of activating dendritic cells and delivering antigens directly into the cytosol for processing and presentation in the MHC class-I pathway. One set of our experiments documents the power of vaccination with DBs in protecting the immunocompetent host against a challenge infection. A further set of experiments revealed a significant improvement of antiviral control in HCT recipients by combining ACT with TherVac. In both settings, the benefit from vaccination with DBs proved to be strictly epitope-specific. The capacity to protect was lost when DBs included the peptide sequence SIINFEKA lacking immunogenicity and antigenicity due to C-terminal residue point mutation L8A, which prevents efficient proteasomal peptide processing and binding to Kb. Our preclinical research data thus provide an argument for using pre-emptive TherVac to enhance antiviral protection by ACT in HCT recipients with diagnosed CMV reactivation.

Immunotherapy of cytomegalovirus infection by low-dose adoptive transfer of antiviral CD8 T cells relies on substantial post-transfer expansion of central memory cells but not effector-memory cells

“…From the very low numbers of viral epitope-specific cells required in AT for control of infection in various organs it becomes intuitively clear that protection is not accomplished by an instant effector function of the few initially transferred cells. Instead, protection must result from the effector function of the progeny cell population generated by in vivo clonal expansion of the transferred cells [33,78]. As we have shown previously [78], as well as here (Fig 3), AT of unseparated memory cells leads to CD8 T-cell tissue infiltrates that are not randomly distributed but accumulate at infected cells in a micro-anatomical structure, the NIF.…”

“…Standard assays for in vivo proliferation are based on loss of a fluorescent reporter dye with every cell division [81,82], but this approach requires AT of high cell numbers, and the resolution is limited to few cell divisions until fluorescence intensity falls below the detection limit. To overcome such technical limitations, we used here our previously described approach of a quantitative ‘input-output’ comparison of the number of initially transferred viral epitope-specific CD8 T cells with the absolute number of progeny cells present in a host organ at defined times after AT [33,78]. CD8 T cells in the stage of cell division were detected in situ by 2C-IHC specific for the CD8 molecule, which localizes to the cytoplasm and cell membrane, and the intra-nuclear ‘proliferating cell nuclear antigen’ PCNA (Fig 7A, images).…”

“…This is easy to explain, as PCNA - tissue-infiltrate CD8 T cells likely have completed proliferation and were thus back to the cell-cycle G0-phase at the time of analysis. Absolute cell counts, extrapolated from tissue sections to the total liver (for the formula, see [33]), revealed an almost absence of iTEM progeny and a strong predominance of TCM progeny over cTEM progeny (Fig 7C), corresponding to integer median cell division numbers (ranges) of 17 (17-18) for TCM, 10 (6-11) for iTEM, and 13 (11-15) for cTEM (Fig 7D).…”

“…Nonetheless, the mouse model has identified basic principles common to all CMVs [21]. As a paramount example for successful clinical translation, mouse models of experimental syngeneic and allogeneic HCT [22–26], for overviews see [27,28], and of control of infection by adoptive transfer (AT) of virus-specific CD8 T cells [29–33] were of predictive value for the management of human infection. Specifically, results from mouse models have paved the way to ‘pre-emptive immunotherapy’ of hCMV infection in HCT recipients.…”

Features of Epstein–Barr Virus and Cytomegalovirus Reactivation in Acute Leukemia Patients After Haplo-HCT With Myeloablative ATG-Containing Conditioning Regimen

“…Furthermore, the pattern of immunoreconstitution was not analyzed due to insufficient data on the dynamic monitoring of lymphocyte subsets. Because our retrospective study focused on recipients after haplo-HCT with an ATG-contained regimen, the impact of serotherapy should be taken into account in further studies ( Lindemans et al., 2015 ; Storek, 2015 ; Willemsen et al., 2015 ; Gergely et al., 2021 ; Keogh et al., 2021 ). Hence, our findings need to be validated by large-scale real-world studies and laboratory investigations in the future.…”