Prospective Monitoring of Polyomavirus BK Replication and Impact of Pre-Emptive Intervention in Pediatric Kidney Recipients

Abstract

Polyoma BK virus (BKV)-associated nephropathy (PVAN) is a relevant cause of poor renal allograft survival.In a prospective analysis, we monitored BKV DNA in blood and urine samples from 62 consecutive pediatric kidney recipients. In patients with BKV replication, we analyzed the impact of reduction of maintenance immunosuppression on viral load kinetics and PVAN in patients with BKV replication. BKV-specific immunity was concomitantly evaluated on blood samples of viremic patients, by measuring the frequency of BKV-specific interferon-c -producing and cytotoxic T cells, and BKV IgG antibody levels. At a median follow-up of 24 months, BK viruria was observed in 39 of 62 patients, while BK viremia developed in 13 patients (21%). In all viremic patients, immunosuppression reduction resulted in the clearance of viremia, and prevented development of PVAN, without increasing the rate of acute rejection or causing graft dysfunction. As a consequence of immunosuppression adjustment, an expansion of BKV-specific cellular immunity was observed that coincided with viral clearance. We conclude that treating pediatric kidney transplant patients pre-emptively with immunosuppression reduction guided by BKV DNA in blood is safe and effective to prevent onset of PVAN. BKV-specific cellular immunity may be useful to guide this intervention.

Characterization of Immunodominant BK Polyomavirus 9mer Epitope T Cell Responses

“…Second, the expansion protocol used in the present study has been devised to overcome the low frequency of BKPyV‐specific T cell responses to overlapping 15mP 35 and was used successfully in the prospective study of pediatric KTRs 30. Our results demonstrate that functional CD8 + T cell responses can be amplified which selectively target few 9mer epitopes located in predicted immunodominant clusters.…”

“…T cell responses to the LVGR‐encoded capsid viral protein VP1 were generally more pronounced than those to EVGR‐encoded viral proteins 30, 35, 49. Interferon γ (IFN‐γ) responses were largely derived from CD4 + T cells and, to a lesser extent, from CD8 + T cells 30, 35, 51, 52, 53. Because most of these studies used overlapping 15mer peptide pools (15mP), the contribution of individual CD8 + T cell–restricted epitopes to these responses is largely undefined.…”

“…We and others investigated cellular immune responses to overlapping peptide pools encoded in the early viral gene region (EVGR) or the late viral gene region (LVGR) of the BKPyV DNA genome 30, 35, 36, 49, 50. T cell responses to the LVGR‐encoded capsid viral protein VP1 were generally more pronounced than those to EVGR‐encoded viral proteins 30, 35, 49.…”

Polyomavirus JC-targeted T-cell therapy for progressive multiple leukoencephalopathy in a hematopoietic cell transplantation recipient

“…On day þ 26, T-cell lines were harvested and examined for immunophenotype, alloreactivity and JCV specificity in a proliferation assay by 3 H-thymidine incorporation, 23 in a cytotoxicity test by standard 51 Crrelease assay, 21 and capacity to produce IFNg in an enzyme-linked immunospot assay. 21 Assays for Ag-specific responses To evaluate JCV-specific responses, patient PBMCs, obtained after density gradient stratification, were cultured for 8 days in the presence of 500 ng/mL JCV-VP1 and LT peptides, and then tested for specific cytotoxicity by means of a standard 51 Cr-release assay against a panel of targets, including autologous PHA blasts pulsed with 2 mg/mL of VP-1 and LT peptide mix or with 2 mg/mL of control peptide. 21 JCV-specific antibodies were tested using viruslike particles, as described previously.…”

“…21 Briefly, 2 Â 10 6 donor PBMCs were pulsed with 15-mer peptide pools, spanning the entire JCV-VP1 and large T (LT) proteins (5 ng/mL concentration of each mix; Eurogentec, Seraing Belgium) 22 in 1 ml volume of X-VIVO 20 medium containing 10% autologous or human AB serum. On day þ 12, cultured cells were recovered, counted and re-plated at 2 Â 10 5 per well in 24-well plates, pre-incubated overnight with 1 Â 10 6 autologous irradiated stimulator PBMC, pulsed with 50 ng/mL JCV-VP1 and LT peptide mixes.…”