Role of BK Virus CTLs in the Treatment of BK Virus-Associated Nephropathy in Kidney-Transplant and Hematopoietic Cell Transplant Recipients.

IntroductionTo better define the biological machinery associated with BK virus (BKV) infection, in kidney transplantation, we performed a proteomics analysis of urinary extracellular vesicles (EVs).MethodsTwenty-nine adult kidney transplant recipients (KTRs) with normal allograft function affected by BKV infection (15 with only viremia, 14 with viruria and viremia) and 15 controls (CTR, KTRs without BKV infection) were enrolled and randomly divided in a training cohort (12 BKV and 6 CTR) used for the mass spectrometry analysis of the EVs (microvesicles and exosomes) protein content and a testing cohort (17 BKV and 9 CTR) used for the biological validation of the proteomic results by ELISA. Bioinformatics and functional analysis revealed that several biological processes were enriched in BKV (including immunity, complement activation, renal fibrosis) and were able to discriminate BKV vs. CTR. Kinase was the only gene ontology annotation term including proteins less abundant in BKV (with SLK being the most significantly down-regulated protein). Non-linear support vector machine (SVM) learning and partial least squares discriminant analysis (PLS-DA) identified 36 proteins (including DNASE2, F12, AGT, CTSH, C4A, C7, FABP4, and BPNT1) able to discriminate the two study groups. The proteomic profile of KTRs with BKV viruria alone vs. viremia and viruria was quite similar. Enzyme-linked immunosorbent assay (ELISA) for SLK, BPNT1 and DNASE2, performed on testing cohort, validated proteomics results.DiscussionsOur pilot study demonstrated, for the first time, that BKV infection, also in the viruric state, can have a negative impact on the allograft and it suggested that, whether possible, an early preventive therapeutic strategy should be undertaken also in KTRs with viruria only. Our results, then, revealed new mechanistic insights into BKV infection and they selected potential biomarkers that should be tested in future studies with larger patients’ cohorts.

Objective:BK virus (BKV) infection is a significant concern for kidney transplantation (KT) recipients, potentially leading to nephropathy and graft loss, particularly under intensive immunosuppression. This study investigates the prevalence of BKV infection and its associated risk factors following KT.Materials and Methods:We conducted a retrospective cohort study on 322 KT recipients undergoing routine follow-up in our unit. BKV infection was defined as either high-level BK viruria (BKV DNA load in urine ≥107 copies/mL) or BKV-associated nephropathy. Risk factors were assessed using univariate and multivariate Cox regression analyses.Results:BKV infection was diagnosed in 9.6% (n=31) of patients, with a median onset of 8.7 months (range: 3.02–31.4). Recipients with BK virus infection were more likely to have received kidneys from non-relative living donors (p=0.005). Smoking and calcineurin inhibitor treatment were more prevalent among infected patients compared to those without BKV infection (p=0.015 and p=0.034, respectively). Additionally, BKV-infected patients experienced higher rates of acute rejection episodes (p=0.009) and all-cause allograft loss (p=0.009). In univariate analysis, smoking (hazard ratio [HR]: 2.697, p=0.007), diabetes mellitus (HR: 2.207, p=0.082), non-relative living donors (HR: 4.355, p=0.001), and induction therapy with anti-thymocyte globulin (ATG) (HR: 2.146, p=0.082) were identified as potential risk factors for infection. Smoking and non-relative living donors were independent risk factors for BKV infection (HR: 2.100, p=0.046 and HR: 4.243, p=0.019, respectively).Conclusion:While immunosuppressive therapy is a well-recognized risk factor for BKV infection, our study highlights smoking and non-relative living donors as independent risk factors. Close monitoring of high-risk recipients and smoking cessation counseling should be prioritized to mitigate BKV infection risk.

A transgenic mouse model was used to identify an HLA-A*02-restricted epitope within the VP1 polypeptide of a human polyomavirus, BK virus (BKV), which is associated with polyomavirus-associated nephropathy in kidney transplant patients. Peptide stimulation of splenocytes from mice immunized with recombinant modified vaccinia virus Ankara expressing BKV VP1 resulted in expansion of cytotoxic T lymphocytes (CTLs) recognizing the sequence LLMWEAVTV corresponding to amino acid residues 108 to 116 (BKV VP1p108). These effector T-cell populations represented functional CTLs as assessed by cytotoxicity and cytokine production and were cross-reactive against antigen-presenting cells pulsed with a peptide corresponding to the previously described JC virus (JCV) VP1 homolog sequence ILMWEAVTL (JCV VP1p100) (I. J. Koralnik et al., J. Immunol. 168:499-504, 2002). A panel of 10 healthy HLA-A*02 human volunteers and two kidney transplant recipients were screened for T-cell immunity to this BK virus VP1 epitope by in vitro stimulation of their peripheral blood mononuclear cells (PBMC) with the BKV VP1p108 peptide, followed by tetramer labeling combined with simultaneous assays to detect intracellular cytokine production and degranulation. PBMC from 4/10 subjects harbored CTL populations that recognized both the BKV VP1p108 and the JCV VP1p100 peptides with comparable efficiencies as measured by tetramer binding, gamma interferon production, and degranulation. CTL responses to the JCV VP1p100 epitope have been associated with prolonged survival in progressive multifocal leukoencephalopathy patients (R. A. Du Pasquier et al., Brain 127:1970-1978, 2004; I. J. Koralnik et al., J. Immunol. 168:499-504, 2002). Given that both human polyomaviruses are resident in a high proportion of healthy individuals and that coinfection occurs (W. A. Knowles et al., J. Med. Virol. 71:115-123, 2003), our findings suggest a reinterpretation of this protective T-cell immunity, suggesting that the same VP1 epitope is recognized in HLA-A*02 persons in response to either BK or JC virus infection.

Correlation between CYP3A5 gene polymorphism and BK virus infection in kidney transplant recipients

Objectives: BK virus (BKV) infection has become one of the main complications in renal transplant recipients (RTRs) with the arrival of newer potent immunosuppressive agents. However, reports on the epidemiology of BKV infection and risk factors in Chinese population after renal transplantation are scarce.Methods: From June 2015 to July 2016, living-donor renal transplant recipients (LDRTRs) who routinely received the quantitative BKV DNA testing of urine and plasma samples using quantitative real-time polymerase chain reaction (PCR) for the first time after transplantation were selected, while dialysis patients and healthy living donors during that period served as controls. Potential variables were compared and analyzed using logistic regression model multivariate analysis to assess the BKV infection related factors in LDRTRs.Results: Among the 52 LDRTRs identified, BKV DNA was detected in 16 urine samples (30.8%), significantly higher than that of dialysis patients (6.3%) and healthy living donors (4.2%) (p < .001). Nevertheless, no statistically significant difference wax noted between the latter two groups in urine samples (p = .842). Meanwhile, BKV DNA detection in blood samples was all negative in the three groups. Univariate analysis shown tacrolimus (Tac) trough level and lymphocyte percentage were associated with BKV infection in LDRTRs. Multivariate regression analysis also showed Tac trough level (HR, 1.644; p = .03), lymphocyte percentage (HR, 0.878; p = .026) were associated with BKV infection in LDRTRs.Conclusions: In Chinese population, the incidence of BKV infection increased significantly after living-donor renal transplantation. Significantly increased Tac trough level and decreased lymphocyte percentage might be the risk factors for BKV infection in LDRTRs.

BK virus (BKV) infection after kidney transplantation can cause BKV nephropathy (BKVAN) resulting in graft dysfunction and allograft loss. The treatment for BKVAN is reduction of the immunosuppressive load which increases the risk of kidney transplant rejection. There is no biomarker to monitor BKV activity besides BK viral load. The value of the Enzyme-Linked Immunosorbent Spot (ELISPOT) assay as a tool to monitor the recipient's anti-BKV immune response after transplantation was investigated systematically. Electronic databases, including MEDLINE, Scopus, and the Cochrane Central Register of Controlled Trials were searched for studies of ELISPOT evaluating the immune response against BKV. BKV status was categorized as "active BKV infection" and as "resolving BKV infection". Random-effects model meta-analysis was performed to determine the diagnostic performance of the ELISPOT assay, after stratifying patients into groups based on positive and negative ELISPOT results. One-hundred twenty-seven articles were identified of which nine were included. Patients with negative ELISPOT had an increased risk of having active BKV replication (odds ratio of 71.9 (95%-CI 31.0-167.1). Pooled sensitivity was 0.95 (95%-CI 0.89-0.98) and specificity was 0.88 (95%-CI 0.78-0.94). The standardized mean difference of the number of IFN-γ producing cells between patients with active BKV infection compared with patients who had resolving BKV infection was -2.09 (95%-CI -2.50, -1.68). The ELISPOT assay is a useful tool for BKV risk assessment and in combination with BKV load may support clinicians in guiding immunosuppressive therapy in patients with BKV replication.

BK virus (BKV) infection and associated BK tubulointerstitial nephropathy (BKVN) still represents a challenge in the renal transplant context (1). Regarding this, molecular tools to test for DNA-BKV are essential in early detection and further management of the kidney transplant recipients (KTR) (2). Although extensive data on this viral infection are now available, some relevant aspects in relation to its pathogenesis, such as donor implication, continue to remain unclear (3). We present new data on BKV infection obtained by a quantitative polymerase chain reaction (PCR) method and a transcription control region (TCR)-BK polymorphism sequence analysis on a renal donor and their corresponding recipient pair. The purpose of the analysis was to determine both the viral genotype involved and the possible role of the renal donor in the origin of this viral infection. Management of BK infection in KTR at the University Hospital La Fe is based on the Nickeleit scheme (4). Since July 2003, a total of 190 KTR have been monitored by protocol. Furthermore, between November 2004 and March 2006, a study was carried out on 58 renal donors and their recipients. This protocol permits us to follow the evolution of those recipient pairs with BK-positive donors that may be at risk of BKV infection. DNA was extracted from pure urine and serum using a QIAamp DNA Virus purification kit for automatic extraction by the BioRobot EZ1 (Qiagen, Valencia, CA). The quantitative PCR method was performed using the SybrGreen QuantiTect PCR assay (Qiagen, Valencia, CA) in the SmartCycler thermocycler (Cepheid, Sunnyvale, CA) to amplify the Large T (LT) and the TCR regions (4). Subsequently, the TCR region was amplified by a cyclesequencing PCR reaction in an automatic sequencer ABI PRISM 3100 (Applied Biosystem, Warrington, UK) using both primers (sense and antisense) examining the polymorphic sites by means of the Basic Local Alignment Search Tool program (http://www.ncbi.nlm.nih.gov) (5). BK infection in KTR could be derived from either the donor or from the transplant recipient (3). Regarding the first possibility, we have found the same JL (Gene Bank: AY628236) viral genotype in a renal donor and their two recipients, supporting an exact identity between the donor and their recipient pair (see Table 1). Thus, we hypothesize that a recipient pair with the same BK genotype might have a possible donor origin of the infection, as was recently reported (3). As Bohl et al., demonstrated, if BKV infection in the recipient originates from the donor kidney, the recipient pairs would be concordant for BKV infection more often than expected (3). In spite of this, Hirsch et al., reported data in favor of the transplant recipient origin of BK infection. They found that 85% of recipients with BKVN were pretransplant seropositive, suggesting that the high-risk group is not the seropositive donor and seronegative recipient transplant combination (6).TABLE 1: DNA-TCR sequence variations examining the four polymorphic sites at the 52–53, 86–92, 145–148 and 179–182 nucleotide position in the donor and their recipient pairIn our opinion, the finding of matching “molecular finger-prints” in both the donor and their recipient pair, supports the hypothesis that posttransplant BKV infection, at least in some cases, derives from the transplant donor kidneys, reinforcing the possible implication of the renal donor as a source of BK infection in KTR. However, larger studies of donors and their recipients are now necessary in order to confirm such observations. Luis A. Rubio Francisco J. Vera-Sempere Service of Pathology Laboratory of Molecular Pathology University Hospital La Fe University Medical School Valencia, Spain

BK virus is a childhood virus that can reactivate in immunocompromised individuals, particularly organ transplant recipients, causing transplant rejection due to BK virus-associated nephropathy. The study aimed to assess the prevalence of BK virus infection in kidney transplant recipients, examine the relationship between demographic and laboratory factors and active infection, evaluate the impact of reducing immunosuppressive drug doses on BK virus reactivation, and explore the genotyping of BK virus strains in this population. This cross-sectional study utilized 245 serum samples from kidney transplant recipients. Viral DNA was extracted from these samples, and initially, Nested PCR was employed for screening to ensure accuracy, with primers targeting a segment of the VP1 gene used to detect the BK virus genome. Real-Time PCR was subsequently performed on positive samples to measure viral load more precisely. The prevalence of BK virus infection among kidney transplant recipients was 5.3%. Out of 245 kidney transplant recipients, 13 individuals were diagnosed with active BK virus infection. Genotype I was the most prevalent, accounting for 90% of the cases. The relationship between demographic factors (gender and age) and laboratory parameters (fasting blood glucose, creatinine, hemoglobin, and platelet count) was examined in both kidney transplant recipients with and without active BK virus infection. The results revealed that a reduction in immunosuppressive drug dosages, particularly tacrolimus, was associated with a decrease in BK viral load, potentially contributing to a lower incidence of active BK virus infections. Additionally, hematological analysis showed a significant decrease in hemoglobin levels in kidney transplant recipients with active BK virus infection, accompanied by a significant increase in serum creatinine levels. Balancing immunosuppressive therapy, especially reducing tacrolimus, helps control BK virus reactivation and preserve graft function. Regular monitoring of hematological parameters and viral load is crucial for optimal management in kidney transplant recipients.

BK Virus Infection in Thai Kidney Transplant Recipients: A Single-Center Experience

Background:BK virus (BKV) belongs to the human Polyomaviridae and the primary BKV infection is occurred during childhood then the virus could be latent through life, especially in the kidneys and urinary system. It became reactive after an immunocompromised status, such as pregnancy or transplantation. Isolated BKV from different locations of the world is grouped into four subtypes using serological and genotyping methods. The BKV subtype I is the dominant one and has worldwide distribution.Objectives:According to our knowledge, there are no data about the BKV prevalence and its genotypes in southwest part of Iran. Considering the high prevalence of renal failure and kidney transplant patients in this part, and the role of BKV in graft rejection, this study aimed to determine the prevalence of BKV infection in renal transplant recipients referred to Golestan Hospital in Ahvaz City, Iran.Patients and Methods:Urine samples were collected from 122 kidney transplant recipients referred to Golestan Hospital in Ahvaz, southwest of Iran. The extracted DNA was amplified by Polymerase Chain Reaction, and subtype of each positive sample was determined using Restriction Fragment Length Polymorphism (RFLP) and sequencing methods.Results;From all study population, 51/122 (41.8%) urine samples were positive for BKV DNA and the other samples were negative (71/122). Forty-eight cases (94.11%) were subtype I and 3 others (5.89%) were subtype IV using the RFLP method. None of the patient’s urine samples were positive for subtypes II and III.Conclusions:Our work is the second study in Iran and considering huge numbers of transplantation in Iran and Khuzestan Province, south western of Iran, in addition to the role of this virus in kidney transplant rejection, routine evaluation of BKV positivity is recommended both for graft recipient and donors. This helps better transplantation result and may prevent graft rejection.

The objective of this study was to characterize CD4(+) and CD8(+) T-cell populations in blood and urine of renal transplant patients with BK virus (BKV) infection or allograft rejection. Percentages and absolute numbers of CD4(+) and CD8(+) effector memory T-cell subtype (TEM ) and terminal differentiated T cells (TTD ) in renal transplant patients with BKV infection (n = 14), with an episode of allograft rejection (n = 9), and in uncomplicated renal transplant patients with a stable kidney function (n = 12) were measured and compared using 4-color fluorescence-activated cell sorting. Results were correlated with the number of CD4(+) and CD8(+) T cells in renal biopsies. In patients with allograft rejection, the number of urinary CD4(+) TEM and CD8(+) TEM cells was significantly increased compared to patients with BKV infection or patients without complications. Positive correlation was found between the number of CD4(+) and CD8(+) cells in the renal biopsies and the number of CD4(+) and CD8(+) cells in urine. In patients with rejection, after 2 months of immunosuppressive therapy, a reduction in urinary CD8(+) TEM cells was found. CD4(+) TEM and CD8(+) TEM cells in urine could be a marker to distinguish allograft rejection from BKV-associated nephropathy and to monitor therapy effectiveness in renal transplant patients with allograft rejection.

Polyomavirus BK virus (BKV) infection represents a serious complication leading to BKV-associated nephropathy (BKVAN) and subsequent kidney graft loss in up to 10% of transplant patients. Cellular immunity is known to play a crucial role in the control of BKV replication. However, the knowledge on the BKV-T-cell response is limited: only two (VP1 and large T antigen) of six known BKV proteins were evaluated for their antigenicity so far. By using 10-color flow cytometry and newly created overlapping peptide pools of five BKV antigens (VP1, VP2, VP3, large T antigen, and small t antigen), we performed cross-sectional phenotypic and functional analysis of BKV-specific T cells in kidney transplant patients with a history of BKVAN. Patients with clinically unapparent BKV infection (history of transient/no BKV reactivation) were used as control group. Our data demonstrate for the first time the antigenic properties of all five evaluated proteins with VP3 as a new important target of cellular immunity. Further, we found a correlation between the severity of the previous BKV infection and the magnitude of memory CD4+ T-cell response. Thus, compared with the control group, patients with a history of BKVAN demonstrated significantly higher frequencies of interferon-γ- and interleukin-2-producing effector memory CD4+ T cells. In the control group, more patients with detectable interferon-γ+/interleukin-2+/tumor necrosis factor+ triple producers were found, suggesting possibly a protective function of these multifunctional T cells. In conclusion, our study results suggest an implementation of new targets for monitoring of BKV immunity. Further studies are required to evaluate the protective function of the found BKV-specific T-cell subsets.

Purpose: BK virus (BKV) infection is a common problem in kidney transplant recipients receiving immunosuppressive therapy, resulting in a serious complications including BKV-associated nephropathy and subsequent allograft loss. The purpose of this study was to characterize the disparate blood subsets in BKV infection/nephropathy at the single-cell transcriptional level. Methods: We isolated peripheral blood mononuclear cells from three kidney transplant recipients with stable status, BK viremia and BK nephropathy. Single-cell libraries were generated using the 10x Genomics Chromium Platform. We analyzed multiplexing data in Cell-Ranger Pipeline and used R and “Seurat” packages for downstream analysis. Results: A total of 14,031 cells were analyzed, including 5473 cells from stable patients, 3976 cells from patients with BK viremia, and 4582 cells from patients with BK nephropathy obtained from Illumina HiSeq X. We characterized 17 distinct clusters representing different cell types. Of these, 13 clusters had differentially expressed transcripts for each sample, and the most differentially expressed markers were S100A8, CCR7, LTB, GNLY, GZMK, GZMH, MT-CO1, LINC02446, IGKC, AIF1, HLA-DRA, STMN1. Stable patient had more mRNA upregulated in B cells compared to patients with BK virus infection. In BK viremia, NK cells and monocytes were downregulated, whereas in BK nephropathy, mRNA expression was high in gamma delta T cells. Conclusions: Our study revealed that BK virus infection/nephropathy induce unique characteristics in lymphocytes, which could be confirmed through single-cell RNA analysis. Further studies are needed to characterize the innate immune cells involved in the progression of BK nephropathy.

BK virus (BKV) infection was studied prospectively in 50 unselected consecutive patients who had undergone kidney transplantation. Infection was monitored for one year after transplantation. Viral DNA in urine (viruria) and plasma (viremia) samples was detected by nested, qualitative polymerase chain reaction. BKV screening data was available for 92% (n = 46) of patients enrolled in the study. Four groups of patients were distinguished: uninfected patients (group 1, n = 30), patients with viruria (group 2, n = 3), patients with viremia (group 3, n = 6) and patients with developed BKV nephropathy (group 4, n = 7). Infection was observed starting form the first month, and the maximum number of patients with active BKV infection occurred at six months after transplantation. Five-year graft survival was 69% for patients with any evidence of BKV infection, compared with 80.0% (P = NS) for patients without BKV infection. The best graft function was observed in group one patient (mean serum creatinine 130 mkmol/l and glomerular filtration rate (GFR) 60.9 ml/min) and the worst in group 4 (mean serum creatinine 180 mkmol/l and GFR 52.31 ml/min) at five years after transplantation. Five-year patient survival was 82.6% and was not affected by presence of BKV infection.

BK virus (BKV) infection is a significant complication in kidney transplant recipients, potentially leading to graft loss. The relationship between pre-transplant tacrolimus (TAC) pharmacokinetics and BKV infection risk remains unclear. This study aimed to investigate whether pre-transplant TAC blood concentration fluctuations are associated with BKV infection risk. We conducted a retrospective study of 135 living donor kidney transplant recipients at Hirosaki University between 2006 and March 2024. Patients were divided into BKV-infected (BKV) and non-infected (non-BKV) groups. TAC blood concentrations were measured at 4 points, including 0 h (2 h before TAC administration), 4, 6, and 12 h on the day before transplantation. Changes in TAC concentration from baseline (0h) were calculated for each time point. The concentration/dose (C0/D) ratio was used as an indicator of TAC metabolism rate. During a median follow-up of 54months, 29 recipients developed BKV infection. The BKV group had significantly older donors and showed a significantly larger decrease in TAC concentration at 12h compared to the non-BKV group (-1.5 vs. 0ng/mL, P = 0.008). There was no significant difference in pre-transplant C0/D ratios between the two groups. A decrease of ≥ 1.5ng/mL at 12h was identified as a significant risk factor for BKV infection (hazard ratio: 2.44, 95% confidence interval: 1.11-5.32, P = 0.026) in a propensity score-based inverse probability of treatment weighting multivariate Cox proportional hazards analysis. Pre-transplant TAC blood concentration fluctuations, particularly a large decrease at 12h from baseline, may be associated with increased BKV infection risk.