Impaired T and "memory-like" NK-cell reconstitution is linked to late-onset HCMV reactivation after letermovir cessation.

Allogeneic hematopoietic stem cell transplantation (alloSCT) is the only cure for many hematologic malignancies. However, alloSCT recipients are susceptible to opportunistic pathogens such as human cytomegalovirus (HCMV). Letermovir prophylaxis has revolutionized HCMV management, but the challenge of late HCMV reactivations has emerged. Immunological surrogates of clinically significant HCMV reactivations (csCMVi) after discontinuation of letermovir remain to be defined. Therefore, we studied NK-cell reconstitution along with the global and HCMV pp65-specific T-cell repertoire of 24 alloSCT recipients at seven timepoints before (day +90) and after (days +120-270) cessation of letermovir prophylaxis. Patients who experienced csCMVi had lower counts of IFNγ+ HCMV-specific CD4+ and CD8+ T cells than HCMV controllers. Furthermore, csCMVi patients displayed late impairment of NK-cell reconstitution, especially suppression of "memory-like" CD159c+CD56dim NK-cell counts that preceded csCMVi events in most patients. Moreover, several surrogates of immune reconstitution were associated with the severity of HCMV manifestation, with patients suffering from HCMV end-organ disease and/or refractory HCMV infection harboring least HCMV-specific T cells and "memory-like" NK cells. Altogether, our findings establish an association of delayed or insufficient proliferation of both HCMV-specific T cells and "memory-like" NK cells with csCMVi and the severity of HCMV manifestations following discontinuation of letermovir prophylaxis.

Identification of novel canonical and cryptic HCMV-specific T-cell epitopes for HLA-A∗03 and HLA-B∗15 via peptide-PRISM.

ABSTRACT: Human cytomegalovirus (HCMV) reactivation poses a substantial risk to patients receiving tranplants. Effective risk stratification and vaccine development is hampered by a lack of HCMV-derived immunogenic peptides in patients with common HLA-A∗03:01 and HLA-B∗15:01 haplotypes. This study aimed to discover novel HCMV immunogenic peptides for these haplotypes by combining ribosome sequencing (Ribo-seq) and mass spectrometry with state-of-the-art computational tools, Peptide-PRISM and Probabilistic Inference of Codon Activities by an EM Algorithm. Furthermore, using machine learning, an algorithm was developed to predict immunogenicity based on translational activity, binding affinity, and peptide localization within small open reading frames to identify the most promising peptides for in vitro validation. Immunogenicity of these peptides was subsequently tested by analyzing peptide-specific T-cell responses of HCMV-seropositive and -seronegative healthy donors as well as patients with transplants. This resulted in the direct identification of 3 canonical and 1 cryptic HLA-A∗03-restricted immunogenic peptides as well as 5 canonical and 1 cryptic HLA-B∗15-restricted immunogenic peptide, with a specific interferon gamma-positive (IFN-γ+)/CD8+ T-cell response of ≥0.02%. High T-cell responses were detected against 2 HLA-A∗03-restricted and 3 HLA-B∗15-restricted canonical peptides with frequencies of up to 8.77% IFN-γ+/CD8+ T cells in patients after allogeneic stem cell transplantation. Therefore, our comprehensive strategy establishes a framework for efficient identification of novel immunogenic peptides from both existing and novel Ribo-seq data sets.

CD4+ T cells are the major predictor of HCMV control in allogeneic stem cell transplant recipients on letermovir prophylaxis.

Introduction: Human cytomegalovirus (HCMV) causes significant morbidity and mortality in allogeneic stem cell transplant (alloSCT) recipients. Recently, antiviral letermovir prophylaxis during the first 100 days after alloSCT replaced PCR-guided preemptive therapy as the primary standard of care for HCMV reactivations. Here, we compared NK-cell and T-cell reconstitution in alloSCT recipients receiving preemptive therapy or letermovir prophylaxis in order to identify potential biomarkers predicting prolonged and symptomatic HCMV reactivation. Methods: To that end, the NK-cell and T-cell repertoire of alloSCT recipients managed with preemptive therapy (n=32) or letermovir prophylaxis (n=24) was characterized by flow cytometry on days +30, +60, +90 and +120 after alloSCT. Additionally, background-corrected HCMV-specific T-helper (CD4+IFNγ+) and cytotoxic (CD8+IFNγ+CD107a+) T cells were quantified after pp65 stimulation. Results: Compared to preemptive therapy, letermovir prophylaxis prevented HCMV reactivation and decreased HCMV peak viral loads until days +120 and +365. Letermovir prophylaxis resulted in decreased T-cell numbers but increased NK-cell numbers. Interestingly, despite the inhibition of HCMV, we found high numbers of "memory-like" (CD56dimFcεRIγ- and/or CD159c+) NK cells and an expansion of HCMV-specific CD4+ and CD8+ T cells in letermovir recipients. We further compared immunological readouts in patients on letermovir prophylaxis with non/short-term HCMV reactivation (NSTR) and prolonged/symptomatic HCMV reactivation (long-term HCMV reactivation, LTR). Median HCMV-specific CD4+ T-cell frequencies were significantly higher in NSTR patients (day +60, 0.35 % vs. 0.00 % CD4+IFNγ+/CD4+ cells, p=0.018) than in patients with LTR, whereas patients with LTR had significantly higher median regulatory T-cell (Treg) frequencies (day +90, 2.2 % vs. 6.2 % CD4+CD25+CD127dim/CD4+ cells, p=0.019). ROC analysis confirmed low HCMV specific CD4+ (AUC on day +60: 0.813, p=0.019) and high Treg frequencies (AUC on day +90: 0.847, p=0.021) as significant predictors of prolonged and symptomatic HCMV reactivation. Discussion: Taken together, letermovir prophylaxis delays HCMV reactivation and alters NK- and T-cell reconstitution. High numbers of HCMV-specific CD4+ T cells and low numbers of Tregs seem to be pivotal to suppress post-alloSCT HCMV reactivation during letermovir prophylaxis. Administration of more advanced immunoassays that include Treg signature cytokines might contribute to the identification of patients at high-risk for long-term and symptomatic HCMV reactivation who might benefit from prolonged administration of letermovir.