Neutralization serotyping of BK polyomavirus infection in kidney transplant recipients

Abstract

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.

Figure 1. Immunization of mice with BKV-I and BKV-IV VLPs.

Six mice were immunized with BKV-I (red circles) or BKV-IV (blue squares) VLPs. In the top panel, sera were serially diluted and tested in separate BKV-I (x axis) or BKV-IV (y axis) VLP ELISAs. A data point from one relatively non-responsive animal is shown as an open circle. The middle panel depicts BKV type-specific neutralizing titers for the same set of mice. The gray diagonal line depicts a theoretical 1∶1 correlation (i.e., perfect cross-reactivity) between BKV-I and BKV-IV titers. The bottom panel shows the ratio of the neutralizing titer for the BKV type administered as a vaccine versus the neutralizing titer for the heterologous BKV type for individual animals. The wide bar represents the geometric mean and the error bars show the 95% confidence interval. Since the BKV-IV ELISA titer of the non-responsive animal could not be calculated, this animal was excluded from the analysis in the bottom panel.

Figure 2. Analysis of sera from healthy adults.

Sera from 48 healthy adults were evaluated for BKV type-specific serological titers. The upper panel shows BKV-I and BKV-IV titers evaluated by ELISA. The lower panel shows neutralizing titers.

Figure 3. BKV-I and BKV-IV serological patterns in kidney transplant recipients.

Sera from kidney transplant recipients were titered for the presence of BKV-I (red circles) or BKV-IV (blue squares) neutralizing antibodies. The neutralizing titer categories shown on the y axis are defined as 1) <95% neutralization at a serum dilution of 1∶100, 2) ≥95% neutralization at 1∶100, 3) ≥95% neutralization at 1∶500, 4) ≥95% neutralizing at 1∶5,000, and 5) ≥95% neutralizing at 1∶50,000. Sera were collected at 5 different time points (x axis) spanning roughly 1, 4, 12, 26, and 52 weeks post-transplantation, designated A-E. In each panel, the notations in the bottom right corner represent the BKV genotype (I or IV) observed in the patient's urine (superscript u) or blood (superscript b). The subject denoted I/IV^u showed urinary shedding of BKV-I at week 5 and urinary shedding of BKV-IV at week 16. The patterns of 12 representative patients are shown. Results for all 108 study subjects are shown in Figure S3.

Figure 4. BKV-I and BKV-IV neutralizing titers in kidney transplant patients at study entry and exit.

Sera from 108 kidney transplant recipients were titered for the presence of BKV-I (top panel) or BKV-IV (bottom panel) type-specific neutralizing antibodies. The percentage of patients at a particular titer cut-off at study entry (1 week after transplantation) are depicted as open bars, while the titers at study exit (1 year after transplantation) are shown as filled bars.