Characterization of the BKPyV-Specific Memory B Cell Response in KTx Recipients and Blood Donors

Background: While BK-Polyomavirus (BKPyV) infection is asymptomatic in immunocompetent individuals, it causes Polyomavirus-associated Nephropathy (PyVAN) in about 10% of all kidney transplant (KTx) recipients. PyVAN is one of the leading causes of premature graft function loss. Importantly, there is currently no virus-specific therapy available. Since the presence of BKPyV-directed antibodies has been associated with control of BKPyV infections, we set out to analyze the memory B-cell response against BKPyV on a single-cell level and isolate BKPyV neutralizing monoclonal antibodies (mAb). Methods: Two cohorts of i.) 200 kidney transplant recipients (KTx) and ii.) 100 blood donors (BD) were screened for plasma neutralization against BKPyV genotypes Ib2 and IV using a pseudovirus neutralization assay. Based on plasma neutralizing activity, we selected the top 5 KTx and 5 BD neutralizers and isolated BKPyV-VP1-specific IgG+ memory B cells from peripheral blood mononuclear cells (PBMC) via Fluorescence activated cell sorting (FACS). Naturally paired heavy and light chain sequences were generated by total RNA reverse transcription and a BCR-specific nested PCR following sequence analysis. Clonality was inferred from IgBLAST annotated sequences and at least one antibody from each identified BKPyV-reactive B cell clone was selected for recombinant antibody production. Monoclonal antibodies were eventually evaluated for VP1-binding and neutralization against BKPyV genotypes (I-IV). Results: Higher neutralization titers and BKPyV cross neutralizing activity against genotypes I-IV was observed in sera from KTX patients compared to the BD group. 1089 BKPyV specific memory B-cells from the top 5 neutralizers from each group were isolated and 768 memory B-cell receptor sequences analyzed. As a first proof of principle, 16 mAbs (5 BD, 11KTx) were produced and screened for neutralizing activity against all genotypes. Of those, mAb COR32_1_C2(KTx) showed a potent cross neutralizing activity (max. IC50 <0.03 ug/ml) against all genotypes. Conclusion: We established a pipeline to isolate BKPyV-Vp1-specific antibodies from human peripheral blood and found BKPyV-neutralizing antibodies in both the BD and the KTX group. Importantly, we identified COR32_1_C2 (KTx) as a promising candidate with highly potent neutralizing activity against all BKPyV genotypes.