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

ABSTRACT: 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 infection (csCMVi) after discontinuation of letermovir remain to be defined. Therefore, we studied natural killer (NK)-cell reconstitution along with the global and HCMV pp65-specific T-cell repertoire of 24 alloSCT recipients at 7 time points before (day +90) and after (days +120-270) cessation of letermovir prophylaxis. Patients who experienced csCMVi had lower counts of IFN-γ+ HCMV-specific CD4+ and CD8+ T cells than HCMV controllers. Furthermore, patients with csCMVi 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 after 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.