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BK polyomavirus (BKV or BKPyV) associated nephropathy affects up to 10% of kidney transplant recipients (KTRs). BKV isolates are categorized into four genotypes. It is currently unclear whether the four genotypes are also serotypes. To address this issue, we developed high-throughput serological assays based on antibody-mediated neutralization of BKV genotype I and IV reporter vectors (pseudoviruses). Neutralization-based testing of sera from mice immunized with BKV-I or BKV-IV virus-like particles (VLPs) or sera from naturally infected human subjects revealed that BKV-I specific serum antibodies are poorly neutralizing against BKV-IV and vice versa. The fact that BKV-I and BKV-IV are distinct serotypes was less evident in traditional VLP-based ELISAs. BKV-I and BKV-IV neutralization assays were used to examine BKV type-specific neutralizing antibody responses in KTRs at various time points after transplantation. At study entry, sera from 5% and 49% of KTRs showed no detectable neutralizing activity for BKV-I or BKV-IV neutralization, respectively. By one year after transplantation, all KTRs were neutralization seropositive for BKV-I, and 43% of the initially BKV-IV seronegative subjects showed evidence of acute seroconversion for BKV-IV neutralization. The results suggest a model in which BKV-IV-specific seroconversion reflects a de novo BKV-IV infection in KTRs who initially lack protective antibody responses capable of neutralizing genotype IV BKVs. If this model is correct, it suggests that pre-vaccinating prospective KTRs with a multivalent VLP-based vaccine against all BKV serotypes, or administration of BKV-neutralizing antibodies, might offer protection against graft loss or dysfunction due to BKV associated nephropathy.

Background Merkel cell carcinoma (MCC) is associated with Merkel cell polyomavirus (MCPyV) infection. MCPyV antibodies (MCPyV-Ab) in plasma correlate with survival, while MCPyV-Ab within the tumor has never been investigated. This study evaluated plasma MCPyV-Ab and tumor MCPyV-Ab titers to evaluate their role in outcomes and prognostication. Methods A single-institution, prospective database was retrospectively reviewed for patients diagnosed with MCC from 2014 to 2021. MCPyV-Ab plasma and tumor titers, as well as patient and treatment factors, were collected. Two-year overall survival (OS) and disease-free survival (DFS) were examined based on MCPyV-Ab presence in tumor. Results Forty patients were identified, with a median follow-up of 27.6 months. Patients were stratified into four groups based on the presence of MCPyV-Ab in plasma (P+, P−) and tumor (T+, T−). Most patients (60.0%) were P−/T−. Of the remaining patients, 22.5% were P+/T+, 12.5% were P−/T+, and 5.0% were P+/T−. Two-year DFS of the P−/T− group was 16.6 months, which was not different from the other groups ( p  = 0.79). Two-year OS of P−/T− was 18.3 months, and 2-year OS of P−/T+ was 28.1 months, which was similar between groups ( p  = 0.80). Conclusions Most patients P+ for MCPyV had antibody-positive tumors (T+), and P− patients were also T−; however, there was a subset of patients where plasma and tumor antibody findings were incongruent. Patients with MCPyV-Ab in either plasma or tumor had a trend toward improved 2-year DFS and OS, but was limited by a small cohort. This study offers an exploratory investigation into the relationship between plasma and tumor antibodies to MCPyV on which to base future work.

Merkel cell polyomavirus (MCPyV) viral protein 1 (VP1) is the capsid protein that mediates virus attachment to host cell receptors and is the major immune target. Given the limited data on MCPyV VP1 mutations, the VP1 genetic variability was examined in 100 plasma and 100 urine samples from 100 HIV+ individuals. Sequencing of VP1 DNA in 17 urine and 17 plasma specimens, simultaneously MCPyV DNA positive, revealed that 27 samples displayed sequences identical to VP1 of MCC350 strain. VP1 from two urine specimens had either Thr47Ser or Ile115Phe substitution, whereas VP1 of one plasma contained Asp69Val and Ser251Phe substitutions plus deletion (∆) of Tyr79. VP1 DNA in the remaining samples had mutations encoding truncated protein. Three-dimensional prediction models revealed that Asp69Val, Ser251Phe, and Ile115Phe caused neutral effects while Thr47Ser and Tyr79∆ produced a deleterious effect reducing VP1 stability. A549 cells infected with urine or plasma samples containing full-length VP1 variants with substitutions, sustained viral DNA replication and VP1 expression. Moreover, medium harvested from these cells was able to infect new A549 cells. In cells infected by samples with truncated VP1, MCPyV replication was hampered. In conclusion, MCPyV strains with unique mutations in the VP1 gene are circulating in HIV+ patients. These strains display altered replication efficiency compared to the MCC350 prototype strain in A549 cells.

Merkel cell polyomavirus (MCPyV) is the foremost causative factor of Merkel cell carcinoma (MCC), a rare yet highly aggressive skin cancer. Although the evaluation of circulating IgG antibodies against Merkel cell polyomavirus (MCPyV) LT/sT oncoproteins is clinically useful for MCC diagnosis/prognosis, a limited number of assays for identifying such antibodies have been developed. Herein, a novel indirect immunoassay with synthetic epitopes/mimotopes of MCPyV oncoproteins was computationally designed and experimentally validated on control sera and sera from healthy individuals and MCC patients. Upon computational design of five synthetic peptides, the performance of the immunoassay in detecting anti‐oncoprotein IgGs in MCPyV‐positive and ‐negative control sera was evaluated. The immunoassay was afterwards extended on sera from healthy individuals, and, for longitudinal analysis, MCC patients. Performance properties such as sensitivity and specificity and positive/negative predictive values were adequate. Receiver‐operating characteristic (ROC) curves indicated that the areas under the curves (AUCs) were within the low/moderately accurate ranges. Immunoassay was repeatable, reproducible and accurate. As expected, the serum anti‐oncoprotein IgG prevalence in healthy individuals was low (2%–5%). Anti‐oncoprotein IgGs slightly increased when MCC patients experienced partial tumour remission and/or stable disease, compared to baseline. Our data indicate that the newly developed immunoassay is reliable for detecting circulating anti‐oncoprotein IgGs both in healthy individuals and MCC patients. In this study, a novel immunoassay for detecting human antibodies against Merkel cell polyomavirus LT and sT oncoproteins was developed. Upon computational design of five synthetic peptides, the immunoassay performance was evaluated in (i) control human sera and (ii) serum samples from healthy individuals and Merkel cell carcinoma patients.

BK polyomavirus-associated nephropathy (BKPyVAN) is a major cause of allograft injury and dysfunction in kidney transplant recipients. Current monitoring tools, including viremia and biopsy, have limitations in sensitivity, invasiveness, and timing. To evaluate donor-derived cell-free DNA (dd-cfDNA) in urine and plasma as a dynamic, noninvasive biomarker for monitoring treatment response and predicting rejection risk in patients with biopsy-proven BKPyVAN. In this prospective cohort study, 25 kidney transplant recipients with biopsy-proved BKPyVAN were enrolled and stratified into two cohorts: conventional immunosuppression reduction (CISR,  = 20) and early immunosuppression reduction (EISR,  = 5). A total of 224 urine and plasma samples were collected before biopsy and at 1, 2, 3, and 6 months post-biopsy. dd-cfDNA levels were quantified and correlated with histological features and clinical outcomes. Urinary dd-cfDNA levels significantly declined in the CISR cohort by month 2 (  < 0.01), preceding changes in creatinine and BKPyV reads. In the EISR cohort, urinary dd-cfDNA levels remained stable, suggesting early therapeutic response. Plasma dd-cfDNA effectively identified acute rejection, with elevations in two CISR patients. Histologic injury patterns, including edema and cast formation, correlated with urinary dd-cfDNA concentrations (  = 0.44-0.51,  < 0.05). Combined urinary and plasma dd-cfDNA measurements are promising for noninvasive, dynamic surveillance of BKPyVAN and rejection risk in kidney transplant recipients. Larger, multicenter studies are warranted to define clinical thresholds and standardize integration into immunosuppression management.

Merkel cell polyomavirus (MCPyV), a small DNA tumor virus, has been detected in Merkel cell carcinoma (MCC) and in normal tissues. Since MCPyV infection occurs in both MCC-affected patients and healthy subjects (HS), innovative immunoassays for detecting antibodies (abs) against MCPyV are required. Herein, sera from HS were analyzed with a novel indirect ELISA using two synthetic peptides mimicking MCPyV capsid protein epitopes of VP1 and VP2. Synthetic peptides were designed to recognize IgGs against MCPyV VP mimotopes using a computer-assisted approach. The assay was set up evaluating its performance in detecting IgGs anti-MCPyV on MCPyV-positive (n=65) and -negative (n=67) control sera. Then, the ELISA was extended to sera (n=548) from HS aged 18-65 yrs old. Age-specific MCPyV-seroprevalence was investigated. Performance evaluation indicated that the assay showed 80% sensitivity, 91% specificity and 83.9% accuracy, with positive and negative predictive values of 94.3% and 71%, respectively. The ratio expected/obtained data agreement was 86%, with a Cohen’s kappa of 0.72. Receiver-operating characteristic (ROC) curves analysis indicated that the areas under the curves (AUCs) for the two peptides were 0.82 and 0.74, respectively. Intra-/inter-run variations were below 9%. The overall prevalence of serum IgGs anti-MCPyV in HS was 62.9% (345/548). Age-specific MCPyV-seroprevalence was 63.1% (82/130), 56.7% (68/120), 64.5% (91/141), and 66.2% (104/157) in HS aged 18-30, 31-40, 41-50 and 51-65 yrs old, respectively (p>0.05). Performance evaluation suggests that our indirect ELISA is reliable in detecting IgGs anti-MCPyV. Our immunological data indicate that MCPyV infection occurs asymptomatically, at a relatively high prevalence, in humans.

The objective of this study was to examine the association between serum tacrolimus trough levels and the detection of BK viruria in kidney transplant recipients. We conducted a retrospective study and included kidney transplant recipients who underwent BK viruria screening during 2018–2021. Serum tacrolimus trough levels, urine BK viral load, and potential risk factors were collected. Statistical analysis was performed to identify the association between the serum tacrolimus trough levels and the detection of BK virus in urine (defined as positive BK viruria), as well as to determine the cumulative incidence and risk factors for BK viruria. Out of 243 recipients, 76 had positive BK viruria. The average serum tacrolimus trough level was significantly higher among the positive BK viruria group compared to the BK viruria-negative group. High HLA mismatch was identified as a risk factor for BK viruria. Approximately half of the recipients with average serum tacrolimus trough levels exceeding 10 ng/mL would develop BK viruria early. This study found an association between the average serum tacrolimus trough level and the detection of BK viruria. High HLA mismatch and an average serum tacrolimus trough level exceeding 10 ng/mL should be regarded as a risk for BK viruria.

BK polyomavirus (BKPyV) and cytomegalovirus (CMV) are the main viral pathogens affecting the graft and recipient outcome after allogenic kidney transplantation. It has recently been found that infection with both viruses has a greater impact on kidney graft function than a single infection. We retrospectively analyzed a cohort of 723 recipients who received kidney transplantation between 2007 and 2015 after living and postmortal donation for differences in risk and outcome parameters regarding BKPyV (DNAemia) and CMV (CMV DNAemia) co-infection compared to sole viremias and to patients without viremia. Of all kidney allograft recipients in our cohort, 8.2% developed co-infection with BKPyV DNAemia and CMV DNAemia, 15.1% showed BKPyV viremia alone and 25.2% sole CMV DNAemia. Acute rejection was closely linked with co-infection (multivariable analysis, p = 0.001). Despite the fact that the estimated glomerular filtration rate of patients with co-infection was noticeably reduced compared to patients with BKV or CMV infection alone, transplant survival and patient survival were not significantly reduced. Co-infection with BKPyV and CMV in kidney transplanted patients is significantly associated with inferior allograft function. Since co-infection is strongly associated with acute rejection, co-infected individuals should be considered a risk collective.

BK virus (BKV) is a significant cause of nephropathy in kidney transplantation. The goal of this study was to characterize the course and source of BKV in kidney transplant recipients. We prospectively collected pretransplant plasma and urine samples from living and deceased kidney donors and performed BKV polymerase chain reaction (PCR) and immunoglobulin G (IgG) testing on pretransplant and serially collected posttransplant samples in kidney transplant recipients. Among deceased donors, 8.1% (17/208) had detectable BKV DNA in urine prior to organ procurement. BK viruria was observed in 15.4% (6/39) of living donors and 8.5% (4/47) of deceased donors of recipients at our institution (P = .50). BKV VP1 sequencing revealed identical virus between donor-recipient pairs to suggest donor transmission of virus. Recipients of BK viruric donors were more likely to develop BK viruria (66.6% vs 7.8%; P < .001) and viremia (66.6% vs 8.9%; P < .001) with a shorter time to onset (log-rank test, P < .001). Though donor BKV IgG titers were higher in recipients who developed BK viremia, pretransplant donor, recipient, and combined donor/recipient serology status was not associated with BK viremia (P = .31, P = .75, and P = .51, respectively). Donor BK viruria is associated with early BK viruria and viremia in kidney transplant recipients. BKV PCR testing of donor urine may be useful in identifying recipients at risk for BKV complications.

To investigate the relationship between neutralization escape and persistent high-level BK polyomavirus replication after kidney transplant (KTx), VP1 sequences were determined by Sanger and next-generation sequencing in longitudinal samples from KTx recipients with persistent high-level viruria (non-controllers) compared to patients who suppressed viruria (controllers). The infectivity and neutralization resistance of representative VP1 mutants were investigated using pseudotype viruses. In all patients, the virus population was initially dominated by wild-type VP1 sequences, then non-synonymous VP1 mutations accumulated over time in non-controllers. BC-loop mutations resulted in reduced infectivity in 293TT cells and conferred neutralization escape from cognate serum in five out of six non-controller patients studied. When taken as a group, non-controller sera were not more susceptible to neutralization escape than controller sera, so serological profiling cannot predict subsequent control of virus replication. However, at an individual level, in three non-controller patients the VP1 variants that emerged exploited specific “holes” in the patient’s humoral response. Persistent high-level BK polyomavirus replication in KTx recipients is therefore associated with the accumulation of VP1 mutations that can confer resistance to neutralization, implying that future BKPyV therapies involving IVIG or monoclonal antibodies may be more effective when used as preventive or pre-emptive, rather than curative, strategies.