Antibody responses to recombinant polyomavirus BK large T and VP1 proteins in young kidney transplant patients

Abstract

BK virus (BKV)-specific immunity is critical for polyomavirus-associated nephropathy, but antibody responses are incompletely defined. We compared the hemagglutination inhibition assay (HIA) with immunoglobulin G enzyme immunoassays (EIA) to BKV proteins expressed in baculovirus-infected insect cells. N-terminal, internal, and C-terminal domains of the BKV large T antigen (BKLT) were fused to glutathione S-transferase (GST), yielding GST-BKLTD1, GST-BKLTD2, and GST-BKLTD3, respectively. The BKV capsid VP1 was expressed as a GST fusion (BKVP1) or as a native VP1 assembled into viruslike particles (BKVLP). We tested 422 sera from 28 healthy donors (HD), 99 dialysis patients (DP; median age, 15 years; range, 3 to 32 years), and 46 age-matched kidney transplant patients (KTP; median age, 15 years; range, 2 to 33 years). In HD, HIA and BKVLP EIA both yielded a 91.7% seroreactivity, whereas all other EIA responses were lower (BKVP1, 83.3%; BKLTD1, 25%; BKLTD2, 29%; BKLTD3, 40%). HIA titers significantly correlated with EIA levels for BKVLP, BKVP1, and BKLTD1 but not for BKLTD2 or BKLTD3, which were barely above the cutoff. In DP, the seroreactivities of HIA, BKVLP, and BKLTD1 were lower than that in HD (63.6%, 86.9%, and 10.1%, respectively) and they had lower titers (P < 0.001). In KTP, seropositivities for BKVLP, BKVP1, and BKLTD1 were 78%, 50%, and 17%, respectively, but anti-BKVLP levels increased significantly in KTP with viruria and viremia, whereas anti-BKLTD1 levels increased after clearing sustained BKV viremia. In conclusion, anti-BKVLP is equivalent to HIA in HD but is more sensitive to determine the BKV serostatus in DP and KTP. In KTP, anti-BKVLP responds to recent BKV viruria and viremia, whereas anti-BKLTD1 may indicate emerging BKV-specific immune control.

FIG. 1.

Recombinant BKV proteins. (A) Schematic representation showing the linear structure of BKLT protein. (B) Purification of BKV VP1 from Sf9 cells by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Ponceau staining of blotting membrane). Lane 1, Sf9 cells; lane 2, Sf9 cells with BKVLP in baculovirus expression vector passage 2; lane 3, Sf9 cells with BKVLP in baculovirus expression vector passage 3; lane 4, CsCl-purified BKVLP. (C) Western blotting of results shown in panel B using polyclonal rabbit anti-BKVP1 (1:2,500) antibody and goat anti-rabbit conjugated to horseradish peroxidase (1:10,000). (D) Electron microscopy and negative staining of BKVLP (magnification, ×35,000).

FIG. 2.

Influence of BKV denaturation on EIA levels. KTP with anti-BKVLP and either without anti-BKVP1 responses (patients 1 and 2) or with anti-BKVP1 responses (patients 3, 4, and 5) were analyzed. +, treatment of indicated BKVLP or BKVP1 with 30 mM DTT, 12.5 mM EDTA, and 12.5 mM EGTA at 55°C for 1 h prior to coating to microtiter plate (see Materials and Methods).

FIG. 3.

BKV-specific antibody titers and viral load in selected KTP. Left Y axes of panels show EIA levels (log OD₄₉₂) for BKVLPs (blue solid circles), BKVP1 (white solid triangles), BKLTD1 (red solid squares), BKLTD2 (black solid squares), and BKLTD3 (black solid squares with white X); right Y axes of panels show viral load (log quantitative PCR [qPCR]) for BKV viruria (graph with yellow background) and BKV viremia (graph with orange background).

FIG. 4.

BKVx-specific antibodies and BKV viral load in plasma and urine of KTP. Antibody levels are shown as OD₄₉₂ values on the right y axis for BKVLP, BKVP1, and BKLTD1; the x axis shows time in months posttransplantation. The KTP are grouped according the BKV DNA detection by PCR in urine (bottom) or in plasma (left) as being negative (Neg.), single-sample positive (Sing. Pos.), and sustained positive (Sus. Pos.), i.e., two or more consecutive samples positive. Box and whisker plots, with the upper and lower boundaries of the box representing 75th and 25th percentiles, respectively, and median value (line in box). Outliers are shown with the open circle, and extreme outliers are shown with a filled star above the whiskers. Numbers of samples are indicated above the blots.