Molecular Characterization of BK Polyomavirus Replication in Allogeneic Hematopoietic Cell Transplantation Patients

Background High-level BK polyomavirus (BKPyV) replication in allogeneic hematopoietic cell transplantation (HCT) predicts failing immune control and BKPyV-associated hemorrhagic cystitis. Methods To identify molecular markers of BKPyV replication and disease, we scrutinized BKPyV DNA-loads in longitudinal urine and plasma pairs from 20 HCT patients using quantitative nucleic acid testing (QNAT), DNase-I treatment prior to QNAT, next-generation sequencing (NGS), and tested cell-mediated immunity. Results We found that larger QNAT amplicons led to under-quantification and false-negatives results (P < .001). DNase-I reduced urine and plasma BKPyV-loads by >90% (P < .001), indicating non-encapsidated BKPyV genomes. DNase-resistant urine BKPyV-loads remained infectious in cell culture. BKPyV genome fragmentation of <= 250 bp impaired NGS coverage of genetic variation using 1000-bp and 5000-bp amplicons. Conversely, 250-bp amplicons captured viral minority variants. We identified genotype-specific and genotype-independent changes in capsid Vp1 or T-antigen predicted to escape from antibody neutralization or cytotoxic CD8 T-cells, respectively. Genotype-specific changes in immunodominant 9mers were associated with reduced or absent CD8 T-cell responses. Thus, failure to control BKPyV replication in HCT Patients may involve insufficient genotype-specific cytotoxic CD8 T-cell responses, potentially predictable by low neutralizing antibodies as well as genotype-independent immune escape. Conclusions Our results provide new insights for patient evaluation and for designing immune protection through neutralizing antibodies, adoptive T-cell therapy, or vaccines. BKPyV genome loads in allogeneic hematopoietic cell transplantation are DNase-I sensitive, nonencapsidated DNA fragments of <= 250 bp. This can impair detection of BKPyV diversity by next-generation sequencing and specifically genotype-associated changes mediating BKPyV immune escape from cytotoxic CD8 T-cell killing.

Automated summary

In this study, the replication of BK polyomavirus (BKPyV) was found to be associated with failing immune control and BKPyV-associated hemorrhagic cystitis in allogeneic hematopoietic cell transplantation (HCT). Molecular markers of BKPyV replication and disease were identified through quantitative nucleic acid testing (QNAT), DNase-I treatment, next-generation sequencing (NGS), and cell-mediated immunity testing. The fragmentation of BKPyV genome below 250 bp was found to impair the detection of BKPyV diversity and genotype-associated changes involved in immune escape.