The BK polyomavirus (BKV) is an opportunistic pathogen of the Polyomaviridae family. After an initial respiratory tract infection with BKV in childhood, which is frequently mild or asymptomatic, the virus persists in a latent state primarily within the renal tubules and epithelial cells of the urinary tract (1–3). Immunosuppressive therapy increases the risk of reactivation in kidney transplant recipients (KTRs) (4, 5). Reactivation of latent BKV in KTRs can lead to BKV-associated nephropathy (BKVAN) and is associated with an increased risk of allograft dysfunction or graft failure in up to 30% of cases (6). Currently, no effective antiviral drugs for BKV infection are available, so the primary strategy for managing the BKV infection is to reduce immunosuppression and restore the immune response against BKV; however, reducing immunosuppressive therapy to limit BKVAN can increase the risk of allograft rejection.

Several posttransplant BKV monitoring strategies have been implemented to improve the management of BKV replication in the immunocompromised host. The most widely used early diagnostic screening method is to periodically measure BKV DNA in the blood and urine up to 1 year after transplantation using quantitative PCR. Despite improvements in screening methods based on regular monitoring of the viral load, the incidence of BKVAN has remained stable over the last few years (5). Therefore, additional indicators to predict the progression of BKV infection are needed for to improve treatment outcomes of KTRs.

Monitoring of BKV-specific humoral and cellular immunity has been considered to assess the risk of BKV infection. However, previous studies have shown that the predictive value of pretransplant BKV-IgG antibodies in KTR is uncertain (2, 6–11). Recently, antibodies against the BKV capsid protein VP1 have been reported to have subtype-specific virus-neutralizing activity in KTRs (12). Some studies have reported that recipient and donor BKV serostatus could be an important predictor of BKV reactivation (2, 13). Since BKV in KTRs may be of donor origin, it is important to assess the donor’s serostatus and BKV titer as risk factors for posttransplant BKV infection (13–15). Recently, Saláková et al. reported that the donor BKV-IgG seroprevalence and antibody level were strongly associated with posttransplant BK viremia and BKVAN in a study of 210 KTRs and 130 donors (16). Finally, they confirmed recommendation by Hirsh and Randhawa that KTRs from seropositive donors should be routinely screened for BKV-DNA in plasma much more frequently than KTRs from seronegative donors (5). Regarding the recipient BKV-IgG, Ginevri et al. suggested that the negative antibody status of the recipient was the most substantial predictor of BKV infection in pediatric KTRs (17). Another study has also highlighted the significant association between the recipient’s seronegative status for BK viremia and the development of BKVAN (18).

Several studies have assessed BKV-specific cellular immunity (CMI) in KT patients, although typically only after transplantation. An increase in BKV-specific T cells has been associated with resolution of BK viremia, and cellular immunity has been reported to play an important protective role in regulating BKV proliferation and progression of BKVAN (6, 8, 11, 19–21). In addition, low pretransplant BKV-specific T cells and posttransplant BKV-CMV unresponsiveness or loss have been reported to be associated with BK viremia and BKVAN (6, 22). Notably, a recent study has underscored the diminished proliferative response of BKV-specific CD8 T cells as a contributing risk factor for BKVAN (23).

Given the importance of pre-transplant immunity in preventing post-transplant BKV infection, screening both humoral and cellular immunity before transplantation may be an effective measure to predict and prevent BKV infection. In the present study, we assessed the BKV-IgG serostatus of kidney donors and recipients, and also measured BKV-specific ELISPOT responses in KTRs prior to transplantation, which had not been previously investigated. The aim of this study was to identify pretransplant risk factors for posttransplant BKV infection by assessing BKV-specific humoral and cellular immunity.

The BK polyomavirus is an opportunistic organism that frequently infects immunosuppressed patients after kidney transplantation, with BKVAN being a notable cause of impaired kidney function that may progress to transplant failure in up to 50% of cases (24, 25). Pretransplant risk factors associated with reactivation of BKV have been reported, including anti-thymoglobulin, administration of enhanced immunosuppressants, history of previous acute rejection, old age, HLA mismatch, HLA-C7 deficiency and anti-BKV IgG positivity (26). In the present study, pretransplant risk factors were evaluated using donor/recipient anti-BKV IgG levels and recipient BKV ELISPOT results to predict post-transplant BKV infection.

The BK virus, which causes infection in KTRs, could theoretically originate from both kidney donors and recipients (27). Anti-BKV IgG seropositivity through the manufacturer’s suggested cut-off levels was 79.6% in KTR and 72.3% in healthy participants, which are similar to the previous study (28). Regarding the association between pretransplant anti-BKV IgG results and posttransplant BKV infection, in a previous study, KTRs who were negative for anti-BKV IgG had four-fold greater incidence of posttransplant BKV infection when the donor was positive for anti-BKV IgG, compared with donors that were negative for anti-BKV IgG (29). Several studies have reported a significantly higher risk of BKV infection in patients transplanted from anti-BKV-IgG-positive donors (14, 30, 31). However, Hirsch et al. suggested that the D+/R− group is not the highest-risk group (28), and the significance of the donor or the recipient anti-BKV antibody serostatus is still uncertain. In the present study, there was a trend toward a greater percentage of KTRs who received allograft from seropositive donors (D+) developing BK viremia, but the pretransplant serostatus of donor and recipients was not significantly associated with posttransplant BK viremia (p >0.05). However, when anti-BKV IgG levels were examined instead of seropositivity, the D-H/R-L group had the greatest risk of developing BK viremia (p <0.001). These findings are consistent with the those of Saláková et al. (16) who established a significant correlation between donor seropositivity and recipient post-transplant BKV infection. A trend toward donor seropositivity and BKVAN was observed in their study, but lacked statistical significance. However, when assessing BKV IgG levels, both post-transplant BKV infection and BKVAN were more prevalent in recipients with higher donor BKV-IgG levels. They also highlighted a significantly increased risk of BKV infection after transplantation in the D+/R- group. In terms of BKVAN, four out of five BKVAN patients received transplants from the D-H group. These results are consistent with previous studies suggesting that the high risk of posttransplant BKV infection is not limited to the D+/R- group and that anti-BKV IgG levels in both donors and recipients may play an important role (28, 30, 32). Our data consistent with those of previous studies indicating that transplantation from donors with high anti-BKV IgG levels (D-H) into recipients with low anti-BKV IgG levels (R-L) is an independent risk factor for BKV infection after transplantation by Dakroub et al. (10, 13). Smith et al. (18) also reported that BKV seronegative in 173 pediatric kidney transplant recipients was strongly associated with the incidence of BKVAN after transplantation. These findings suggest the importance of standardized anti-BKV-IgG testing and of clear guidelines for evaluation of anti-BKV-specific IgG levels and the definition of seropositivity. Currently, pretransplant screening of anti-BKV serostatus in kidney donors and recipients is neither mandatory nor routinely performed. Further studies are required to investigate the relationships between posttransplant BK viremia and pretransplant anti-BKV IgG antibody levels measured using standardized assays.

In terms of cellular immunity, BKV-specific T cells play an important role in the control of BKV infection by maintaining a persistent memory T cell response to the virus (19, 33–35). A prior meta-analysis reported that ELISPOT assays could be an effective tool to assess BKV-specific cellular immunity (7). In previous studies, posttransplant monitoring of BKV-specific cellular immune responses were conducted to investigate their associations with posttransplant reactivation of BKV (34, 36, 37). However, there have been few studies assessing whether pretransplant BKV-specific immunity levels can predict posttransplant BKV reactivation. Mutlu et al. (8) analyzed BKV-specific CD4+ T-cell responses of KTRs before transplantation and did not show any association between pre-transplant response and BKV reactivation, like the other two previous studies (6, 38). However, they confirmed that clearance of BK viremia or decrease of viral load corresponds with an increase in BKV-specificCD4+T-cellresponse. Other reports have also suggested that the absence of or low response of BKV-specific T cells may be a risk factor for BKV reactivation, while others have found no clear association (6, 8, 39). Dekeyser et al. found a gradual loss of BKV-specific T-cell polyfunctionality with BKV reactivation, particularly in patients with BKVAN. This exhaustion of BKV-specific T-cell responses is linked to impaired control of viral replication in the kidney (23). In present study, KTRs tended to have greater and broader BKV-ELISPOT results before transplantation compared to healthy controls, with a statistically significant difference for VP2-ELISPOT. Next, we investigated whether the pretransplant BKV-ELISPOT results could predict the development of posttransplant BK viremia. In our study, the 14 BK viremia (+) patients tended to have lower pretransplant ELISPOT responses to all 5 BKV peptides than BK viremia (-) patients. Among the five peptide-specific ELISPOTs, significant differences were observed for the St, VP3 and total BKV-ELISPOT results. The ROC analysis of total BKV-ELISPOT results showed an AUC of 0.686 and sensitivity of 71.4% and specificity of 54.4% using the cut-off value of ≤53 spots/300,000 cells. Also, four of five BKVAN patients had decreased pretransplant BKV-ELISPOT results. Our data are consistent with those of a previous report suggesting that specific T-cells control BKV latency before transplantation, and in this way may influence BKV reactivation after transplantation (40). These results suggest that low BKV-specific cellular responses before transplantation can be used as a risk factor to predict the development of BK viremia after transplantation. Regarding the target antigens of BKV-specific cellular immunity, our data did not identify the dominant target antigen. This finding confirms previous reports that it is not clear whether certain BKV peptides have a more dominant antigenicity (36, 41).

Given both BKV-IgG levels and BKV-ELISPOT results were correlated with BK viremia (> 10⁴ copies/mL of BKV-DNA detected during the follow-up period), we analyzed the diagnostic performance of a combination of these biomarkers. Among KTRs who exhibited low levels of anti-BKV-IgG and low total BKV-ELISPOT results, and who received a kidney from the D-H group, the PPV and NPV for BKV viremia after transplantation were found to be 41.7% and 88.9% respectively. The relationship between impairment of BKV-specific cellular immunity and increased BKV replication in BK viremia remains uncertain (39). In a previous study, impaired functionality of cytomegalovirus (CMV) or BKV-specific T cells was evident in some transplant recipients for a prolonged period even after virus control (42–44). In present study, we used threshold of 10⁴ copies/mL for BK viremia and initiation of therapeutic intervention. However, several studies have explored alternative thresholds for initiating therapy to prevent the progression to BKVAN. For instance, Hassan et al. reported that the currently suggested plasma viral load cutoff of 10⁴ copies/mL for BK virus tends to underestimate the diagnosis of BKVAN (45). Another study by Hirsch et al. also indicated that viral nephropathy was linked to plasma viral loads of 7.7×10³ copies/mL or higher (28). Randhawa et al. emphasized customized thresholds for medical centers, given their significant impact on predictive BKV infection outcomes (46). When we reexamined the dataset using a threshold of 10³ copies/mL for BK viremia, two additional patients were reclassified into the BK viremia group. Consequently, the association between donor BKV-IgG levels and BK viremia (> 10³ copies/mL) after KT was also significant (P value = 0.001). Similarly, BKV-specific ELISPOT results showed comparable diagnostic performances between data using thresholds of 10³ copies/mL and 10⁴ copies/mL ( Supplemental Table 1 ). It is very important to establish risk factors for BKV infection after transplantation and application of proper BKV viral load cutoff, because careful monitoring of serum BKV and early reduction of immunosuppressants can resolve BK viremia and prevent transplant failure due to BKVAN in transplanted kidneys (47). Close monitoring after transplantation is necessary for patients whose donors showed high absorbance before kidney transplantation. Our results indicate anti-BKV-antibody-negative patients with a low ELISPOT results are expected to be at greater risk of BKV reactivation, so thorough monitoring is required. Thus, the application of pretransplant screening tests using anti-BKV-IgG and ELISPOT assays in conjunction with assessment of posttransplant blood BKV-DNA viral load may provide more accurate guidance for therapeutic intervention in KTRs with BKV infection.

This study had some limitations. First, a prospective cohort analysis was conducted at a single center including relatively small number of patients. Second, only five of 14 BK viremia (+) patients developed BKVAN. Therefore, our study could not determine whether pretransplant anti-BKV-IgG and BK-ELISPOT results can identify KTRs who will develop BKVAN. Finally, anti-BKV-IgG levels were not validated in this study. For clinical application, cut-off values should be investigated with standard methods to stratify individual risk of BKV infection. In addition, our study used a commercially available anti-BKV IgG kit targeting the BKV serotype I and IV genotypes. Therefore, applying our results to other BKV serotypes could potentially introduce variability. Follow-up investigations focusing on all BKV serotypes are essential to comprehensively assess clinical significance of BKV serostatus. Well-designed prospective studies controlling for relevant confounding factors are needed before using anti-BKV-IgG and BKV-ELISPOT assays clinically to predict posttransplant BKV infection. Despite these limitations, our study focused on BKV serostatus and BKV-ELISPOT results before transplantation and demonstrated the diagnostic value of combining these tests. In conclusion, pretransplant donor and recipient anti-BKV-IgG levels and BKV-ELISPOT assay results may be used to identify patients at risk of BKV infection. Further studies are needed to confirm our results and validate the anti-BKV-IgG cut-off levels and BKV-ELISPOT assays as tools to predict post-kidney-transplant BKV infection.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving humans were approved by Institutional Review Board at Seoul St. Mary’s Hospital (KC19TESI0043). The studies were conducted in accordance with the local legislation and institutional requirements. The human samples used in this study were acquired from a by- product of routine care or industry. Written informed consent for participation was not required from the participants or the participants’ legal guardians/next of kin in accordance with the national legislation and institutional requirements.

HB performed the experiments and wrote the paper. HB and SJ analyzed the data. E-JO designed the study and edited the paper. BC and CY supervised specimen selection and the collection of clinical information. All authors contributed to the article and approved the submitted version.