Sixty-eight KTRs, who agreed to donate peripheral blood at the time of measuring BKV-DNA using RT-qPCR, were included in this study. All participants provided written informed consent in accordance with the Declaration of Helsinki. This study was approved by the Institutional Review Board at Seoul St. Mary’s Hospital (KC16TISI0700). They were divided into three groups according to their viremia status before and at enrollment, evaluated by routine screening for BKV in our transplant center [24]; (1) no viremia (n = 17), (2) BK viremia (n = 27), and (3) cleared viremia (n = 24). The no viremia group consisted of patients with no evidence of viral replication at post-transplant routine screening using blood BKV-DNA PCR and for the next 6 months after enrollment. The BK viremia group was comprised of patients who showed significant blood BKV-DNA at the time of the BKV-ELISPOT test, irrespective of results before enrollment. The significant blood BKV-DNA was defined as the value higher than 4 log copies/mL according to our own and also other group’s previous studies [24,25]. In the BK viremia group, we performed subgroup analysis according to the clinical course of BKV infection; the controller group who showed clearance of BK viremia within 3 months, and the noncontroller group, who had persistent viremia up to 6 months after diagnosis of BKV infection (Fig. 1) [26]. Clearance of BK viremia indicates that blood BKV PCR is less than 500 copies/mL. The cleared viremia group included patients who had no viremia at the time of enrollment, but experienced viremia previously. We also included 44 HCs.

BKV-DNA quantification in whole blood was performed by RT TaqMan PCR kit (Applied Biosystems, Foster City, CA, USA) to detect target viral capsid protein (VP-1) gene using an ABI PRISM 7000 real-time PCR system (Applied Biosystems). We performed BKV monitoring and preemptive immune suppressant reduction according to our transplant center’s protocol [24]. Briefly, plasma BKV PCR was performed at 1, 3, 6, 9, 12 months after transplantation, and once a year afterward or when allograft dysfunction was detected. We defined significant BK viremia as detection of BKV (> 4 log copies/mL) in the blood sample [24,25]. When significant BK viremia was detected, we discontinued MMF and monitored BKV RT-qPCR every month until BK viremia disappeared. When BK viremia was sustained after one month, we performed allograft biopsy, and the dose of calcineurin inhibitor (CNI) was reduced by 20%, and also started leflunomide [24]. Diagnosis of BKVN was confirmed when the following criteria were met: (1) typical histological features suggestive of BKVN such as presence of intra-nuclear viral inclusions, (2) positive SV40T by immunoperoxidase staining, and (3) detection of BKV replication in at least one test among urine cytology, urine PCR, or plasma PCR [27].

BK virus has double-stranded DNA genomes which include two early enzymatic proteins and three late structural proteins. The early proteins are the large tumor antigen (LT) and the small tumor antigen (St). These T antigens are responsible for cell immortalization and latency. Late structural proteins consist of viral capsid: VP1, VP2, and VP3 [28,29]. We measured T cell responses to each of the five proteins using ELISPOT assay. We used a commercial (Human interferon γ [IFN-γ] ELISPOT Ready-SET-Go, eBioscience, San Diego, CA, USA), 96-well ELISPOT plates (Millipore, Cat. No. MAIPS4510) were washed and coated overnight at 4°C with 100 μL/well of a mouse monoclonal anti-human IFN-γ capture antibody diluted in ELISPOT coating buffer (reconstitute powder to 1 L in dH2O). And then, coating antibody was aspirated from plate. Plates were washed with 200 μL/well of sterile ELISPOT coating buffer and blocked with 200 μL/well of complete RPMI-1640 (with Fetal Bovine Serum and 1% Penicillin/Streptomycin/L-Gutamine) at room temperature for 1 hour. Peripheral blood mononuclear cells (PBMCs) (3 × 105 cells/well) were stimulated with phorbol 12-myristate 13-acetate (PMA)/ionomycin (positive control), complete media (negative control) and BKV peptide (1 μg/mL of VP1, VP2, VP3, LT, and St, JPT, Peptides Technologies, Berlin, Germany) diluted in complete RPMI-1640 to appropriate wells at 100 μL/well in 5% CO₂ humidified incubator, at 37°C for 24 hours. Plates were washed with ELISPOT wash buffer (1 × PBS, with 0.05% Tween-20) and diluted by biotinylated detection antibody (Anti-Human IFN gamma Biotin) in Assay Diluent (1X in dH₂O) with 100 μL/well of detection antibody solution. Incubate at room temperature for 2 hours. Next, plates were washed with ELISPOT wash buffer, diluted by Avidin-HRP reagent in Assay Diluent with 100 μL/well of Avidin-HRP solution and incubate at room temperature for 45 minutes. Then, washed with ELISPOT wash buffer and two times with 1 × PBS (no Tween-20). Plates were developed at room temperature for 30 minutes with 100 μL/well of freshly-prepared AEC (3-amino-9-ethyl carbazole) substrate solution and stopped the substrate reaction by washing well with 200 μL/well of distilled water. Air-drying the plate, resulting spots were counted using a computer-assisted ELISPOT image analyzer (Cellular Technologies Ltd., Cleveland, OH, USA). Results were calculated as mean values of spots/3 × 10⁵ cells PBMC based on duplicate or triplicate measurements after subtracting the response of negative control wells.

First, we compared results of BKV-ELISPOT among the four groups (HCs [n = 44] and the three KTRs: no viremia [n = 17], BK viremia [n = 27], cleared viremia [n = 24]). Second, we investigated if the BKV-ELISPOT results can predict clearance of BKV infection in the BK viremia group. We compared ELISPOT results at diagnosis of BK viremia between of the controller (n = 10) and the noncontroller (n = 17) groups. In the BK viremia group, 11 patients were diagnosed as BKVN, and we compared ELISPOT results between the BKVN group and no BKVN group.

Statistical analyses were performed using the SPSS software version 24.0 (IBM Co., Armonk, NY, USA). Categorical variables were compared by Pearson’s chisquare test or Fisher’s exact test. Continuous variables were compared using Student’s t test or Mann-Whitney U test between two groups, and we used one-way analysis of variance test or Kruskal-Wallis test to compare continuous variables among KTRs according to viremia status. For non-parametric variables, transformation to ranking variables was used. The relationship between two parameters was assessed by Spearman’s correlation rank test. All tests were two-tailed, and the results were considered significant at p < 0.05.

Table 1 shows the characteristics of the KTRs in this study. Mean age of the KTRs was 50.4 ± 13.5 years and 66% of the study group was male. Comparing KTRs with different viremia status, age of recipients, human leukocyte antigen (HLA) mismatch number and serum creatinine levels were higher in the BK viremia group compared to those in the no viremia group (p < 0.05). There was no difference in percentage of deceased donor-KTs and the induction therapy with anti-thymocyte globulin (ATG) among the three groups (p > 0.05). Serum tacrolimus trough level at the time of BKV-ELISPOT assays, time interval from kidney transplantation to BKV-ELISPOT assays and peak BKV PCR titer in blood were higher in the BK viremia group compared to the cleared viremia group. The BK viremia group patients had received triple immunosuppressive therapy consisting of a tacrolimus, mycophenolic acid (MPA), and prednisolone (PD) (n = 26, 96.2%) before onset of the viremia. Patients who diagnosed with BK viremia changed their maintenance immunosuppressant as followings, MPA withdrawal (n = 6), leflunomide addition (n = 2), MPA withdrawal with leflunomide addition (n = 17), and MPA withdrawal with tacrolimus reduction (n = 2) (Table 2).

KTRs had higher BKV-ELISPOT results against all five peptides than HCs (p < 0.05). All three KTRs group showed significantly higher or higher tendency of ELISPOT results for all five peptides than HCs. Among KTRs with different viremia statuses, we found increasing tendency of ELISPOT results in the no viremia or the cleared viremia group in comparison with the BK viremia group. The BK viremia group had a lower number of spots than the no viremia group but it is not significant. In comparison between the BK viremia and the cleared viremia group, VP1 and VP2 ELISPOT results were significantly higher in the cleared group (p = 0.0434 and p = 0.0245, respectively) (Fig. 2). Between the no viremia and the cleared viremia group, no significant difference was detected for all five peptides.

In the BK viremia group, 10 (37.0%) patients succeeded in BK viral clearance (the controller group) within 3 months, and 17 (63.0%) patients showed persistent viremia for more than 3 months (the noncontroller group). In the noncontroller group, LT-ELISPOT results were significantly lower (p = 0.033) and other ELISPOT results also showed lower tendency compared to the controller group. The controller group had higher ELISPOT SUM which means the sum of each spot count of five BKV peptides (LT, St, VP1, VP2, and VP3) than the nonconatroller group (p = 0.0486) (Fig. 3). We also investigated if BKV-ELISPOT results can predict the development of BKVN. Of 27 KTRs in the BK viremia group, 22 KTRs received allograft biopsies during 6 months’ follow-up, and 11 patients were confirmed as BKVN. The 11 BKVN (+) patients had lower BKV-ELISPOT results for LT, St, VP1, VP2 peptides at the time of BK viremia development than 16 BKVN (–) patients in the BK viremia group (p = 0.036, p = 0.043, p = 0.006, and p = 0.025, respectively). ELISPOT SUM at the time of BK viremia was lower in patients with BKVN than those without BKVN (p = 0.0136) (Fig. 4).