Neutralizing Antibody–Mediated Response and Risk of BK Virus–Associated Nephropathy

CLINICAL RESEARCH www.jasn.org

Morgane Solis,1,2 Aurélie Velay,1,2 Raphaël Porcher,3 Pilar Domingo-Calap,2 Eric Soulier,2 Mélanie Joly,2,4 Mariam Meddeb,1 Wallys Kack-Kack,1 Bruno Moulin,2,4 Siamak Bahram,2 Françoise Stoll-Keller,1,2 Heidi Barth,1,2 Sophie Caillard,2,4 and Samira Faﬁ-Kremer1,2

1Virology Laboratory and 4Nephrology Department, Strasbourg University Hospitals, Strasbourg, France; 2Unité Mixte de Recherche 1109, Institut National de la Santé et de la Recherche Médicale, Strasbourg University, Strasbourg, France; and 3Clinical Epidemiology Center, Centre de Recherche Épidémiologie et Statistique Sorbonne Paris Cité (CRESS), Unité Mixte de Recherche 1153, Institut National de la Santé et de la Recherche Médicale, Paris Descartes University, Paris, France

ABSTRACT BK virus–associated nephropathy (BKVAN) causes renal allograft dysfunction. The current management of BKVAN relies on pre-emptive adaptation of immunosuppression according to viral load monitoring. However, this empiric strategy is not always successful. Therefore, pretransplant predictive markers are needed. In a prospective longitudinal study, we enrolled 168 kidney transplant recipients and 69 matched donors. To assess the value of BKV genotype–specific neutralizing antibody (NAb) titers as a predictive marker for BKV replication, we measured BKV DNA load and NAb titers at transplant and followed patients for 24 months. After transplant, 52 (31%) patients displayed BKV replication: 24 (46%) patients were viruric and 28 (54%) patients were viremic, including 13 with biopsy-confirmed BKVAN. At any time, patients with high NAb titers against the replicating strain had a lower risk of developing BKV viremia (hazard ratio [HR], 0.44; 95% confidence interval [95% CI], 0.26 to 0.73; P=0.002). Each log10 increase in NAb titer decreased the risk of developing viremia by 56%. Replicating strains were consistent with donor transmission in 95% of cases of early BKV replication. Genotype mismatch between recipients’ neutralization profiles before transplant and their subsequently replicating strain significantly increased the risk of developing viremia (HR, 2.27; 95% CI, 1.06 to 4.88; P=0.04). A NAb titer against the donor’s strain ,4log10 before transplant significantly associated with BKV replication after transplant (HR, 1.88; 95% CI, 1.06 to 3.45; P=0.03). BKV genotype–specific NAb titers may be a meaningful predictive marker that allows patient stratification by BKV disease risk before and after transplant.

J Am Soc Nephrol 29: 326–334, 2018. doi: https://doi.org/10.1681/ASN.2017050532

The emergence of BKvirus–associated nephropathy Significance Statement (BKVAN) is one of the major causes of graft dysfunction and loss in kidney transplant recipients. BK virus-associated nephropathy (BKVAN) is one of the BKVAN essentially arises from the use of highly major causes of graft dysfunction and loss in kidney potent immunosuppressive drugs1–4 and is a grow- transplantation. The management of BKVAN is challenging,partlydueto thelack of predictive markers. This ing clinical problem as the population of transplant manuscript describes the discovery of BKV genotype- specific neutralizing antibody titers as a new predictive marker for BKVAN. We demonstrate that recipients who Received May 17, 2017. Accepted September 4, 2017. develop weak antibody neutralizing responses are at a significantly higher risk of developing BKVAN. In addi- Published online ahead of print. Publication date available at tion, we identified a cutoff value that allows patient www.jasn.org. stratification into lower and higher BKV disease risk groups at the time of transplantation. Our findings offer Correspondence: Prof. Samira Fafi-Kremer, Institut de Virologie, 3 Rue Koeberlé, F-67000 Strasbourg, France. Email: samira.fafi- a useful tool for clinicians to improve kidney transplant [email protected] management and pave the way for the development of new preventive or therapeutic strategies. Copyright © 2018 by the American Society of Nephrology

326 ISSN : 1046-6673/2901-326 J Am Soc Nephrol 29: 326–334, 2018 www.jasn.org CLINICAL RESEARCH

recipients continues to increase. BKV replication occurs in BKVAN (medians 5.87, 6.51, and 5.37 log10 copies per millili- 30%–50% of recipients with progression to BKVAN in 7%– ter, respectively; P=0.61). No significant difference was ob- 8% of patients which ultimately leads to graft dysfunction and served for initial viremia between patients who were viremic 5 loss in a majority of BKVAN cases. Furthermore, early BKV and those with BKVAN (medians 4.85 and 4.80 log10 copies per replication after transplantation increases the risk of late milliliter; P=0.80). Sequencing analysis of BKV VP1 identified acute rejection.6 At present, there are no BKV-specificanti- genotype I, genotype IV, and genotype II in 45 (86%), six viral therapies available. Current guidelines recommend regular (12%), and one (2%) patients who were BKV DNA–positive, screening to detect BKV viruria or viremia after renal transplan- respectively (Figure 1B). No significant difference was observed tation.2,7 Patients exhibiting persistently high BKV DNA loads in BKV genotype prevalence among viruric, viremic, and usually undergo immunosuppression reduction to reduce BKV BKVAN groups (P=0.47). No significant difference relative to replication and to prevent BKVAN occurrence. However, be- patients’ attributes or immunosuppressive treatment at cause of its delayed nature, this pre-emptive strategy does not transplantation was noted between the four groups, except fully eliminate the risk of BKVAN8,9 and can increase the risk of for rituximab administration (Supplemental Table 1). Three donor-specific antibodies (DSA), graft rejection, and death.6,10 out of five patients having received rituximab before trans- Therefore, a predictive marker for BKV replication before transplantation developed BKVAN. Acute rejection occurred in plantation is needed to identify patients at risk. Neutralizing 20% (23 of 116), 17% (4 of 24), 26% (4 of 15), and 46% (6 antibodies (NAbs) have been shown to significantly influence of 13) of the patients belonging to the BKV DNA–negative, the outcome of chronic viral infections in patients with impaired viruric, viremic, and BKVAN groups, respectively. Of note, 10 cellular immune responses such as hepatitis C virus–infected of 14 (71%) of the rejection episodes occurred after the di- liver transplant recipients or patients with HIV.11–13 In this pro- agnosisofBKVinfection(2of4forpatientswhowereviruric, spective longitudinal study, for the first time, we analyzed the 3 of 4 for patients who were viremic, and 5 of 6 for the effect of NAbs on the outcome of BKV infection after kidney BKVAN group). During follow-up, seven patients died, two transplantation over a period of 24 months and investigated of whom were BKV DNA–positive. Graft loss occurred in neutralization genotype–specific titers as a predictive marker eight cases after antibody-mediated or cellular rejection in for BKV replication. three and five cases, respectively. Among them, one patient had developed BKVAN.

RESULTS BKV Genotype–Specific Seroconversion after Transplantation Patients’ Characteristics At the time of transplantation, 158 patients (94%) harbored One hundred sixty-eight patients were prospectively followed NAbs against at least one BKV genotype; ten patients were for a mean duration of 6936125 days. BKV load was measured BKV-seronegative. Regarding patients who were BKV DNA– in 2850 pairs of blood and urine samples. NAb titers were negative (n=116), 70 (61%) harbored NAbs against BKV ge- tested in a total of 773 blood samples. Patients’ characteristics notype I, 12 (10%) against genotype II, and five (4%) against are summarized in Table 1. Among the 168 patients, 116 genotype IV at the time of transplantation. Nineteen (16%) (69%) showed no detectable levels of BKV viruria or viremia patients harbored NAbs against two or three BKV genotypes during the time of the study (BKV DNA–negative). Fifty-two and ten (9%) were BKV-seronegative (Figure 2A). At month 24, (31%) patients displayed evidence of BKV replication (BKV BKV genotype neutralization profiles remained unchanged. DNA–positive). Of these, 24 (46%) remained only viruric Patients who were BKV DNA–positive (n=52) showed ge- (viruric group) whereas 28 (54%) developed viremia, includ- notype-specific NAb prevalence rates similar to patients who ing 13 with biopsy-confirmed nephropathy, 7 stage A, 6 stage B, were BKV DNA–negative at the time of transplantation, with and 0 stage C (BKVAN group) (Figure 1A). Initial viruria was 58%, 13%, and 2% of the patients harboring NAbs against similar for patients who were viruric, viremic, and those with genotypes I, II, or IV, respectively. Fourteen (27%) patients harbored NAbs against two or three genotypes. An important Table 1. Patients’ characteristics change in BKV genotype–specific NAb distribution was ob- – Variable Patient Cohort (n=168) served for patients who were BKV DNA positive at month 24, Age, median (range) 56.2 (17.7–75.7) among which 92% displayed NAbs against two or three geno- Male, n (%) 101 (60.1) types, with higher titers directed against the replicating strain First graft, n (%) 128 (79.2) as compared with other strains (P,0.001) (Figure, 2B and C). Living donor, n (%) 24 (14.3) These patients did not display PCR nor sequencing data sug- HLA mismatches, median (range) 4.0 (0.0–6.0) gestive of mixed infection (data not shown). Sixty out of the Cold ischemia time, min, median (range) 841 (70–1861) 69 (87%) analyzed donors harbored NAbs against at least one Thymoglobulin, n (%) 82 (48.8) BKV genotype (62% genotype I, 6% genotype II, 4% genotype Tacrolimus, n (%) 91 (54.2) IV, 9% two genotypes, and 6% three genotypes; Supplemental Rituximab, n (%) 5 (3.0) Figure 1).

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increase of 1.21 (0.84 to 1.72) (Figure 3C, Supplemental Table 2). We observed no association between NAb titers and the AUC of mycophenolate mofetil measured at month 1 (r=20.12, P=0.13; Figure 3D). Four out of ﬁve patients who received rituximab became viremic and showed a delayed increase in NAb titers during follow-up, and three of them developed BKVAN.

Neutralizing Titers before Transplantation Predict the Development and Outcome of BKV Infection after Transplantation To investigate whether pretransplantation NAb titers could predict the occurrence of BKV replication, we compared NAb titers – Figure 1. Monitoring of BK virus (BKV) replication. Kaplan Meier estimates of BKV measured at day 0 in patients who were BKV viruria, viremia, and BKVAN in 168 kidney transplant recipients followed-up for 24 DNA–negative versus those who were BKV months post-transplant. BKVAN was diagnosed on the basis of histologic findings in DNA–positive. NAbs directed against the allograft biopsy specimens. (A) Cumulative incidence values at month 24 are in- replicating BKV genotype were considered dicated. (B) Virologic characteristics of viruric, viremic, and BKVAN groups are shown. – NA, not applicable. for patients who were BKV DNA positive. Asignificant difference was observed between the two groups, with median NAb titers of A Delayed and Weak NAb Response Is Associated with 4.40 log10 IC50 and 3.92 log10 IC50 (P=0.04) in patients who Severe BKV Infection Outcome were BKV DNA–negative and BKV DNA–positive, respectively. Longitudinal measures of NAb titers directed against the rep- This difference suggests that viral replication correlates with ab- licating BKV genotype were performed in patients who were sent or insufficient NAbs against the donor strain. On the other BKV DNA–positive. Four out of 24 (17%) patients who were hand, it appears that patients who were BKV DNA–negative either viruric, 5 of 15 (30%) who were viremic, and 7 of 13 (54%) had sufficient NAb titers to control the replication of any BKV patients with BKVAN displayed a genotype mismatch for their strain “hitchhiking” in the donated organ or that their donors replicating strain. An increase in NAb titers was observed as were BKV-seronegative. early as 1 month after viruria; however, titers and kinetics of We addressed this hypothesis by analyzing sera from 69 the neutralizing response differed among patients (Figure 3, donor/recipient pairs. The analysis of 46 matched sera of A and B and a detailed description of BKV DNA load and NAb donor/BKV DNA–negative recipient pairs at day 0 showed titer kinetics for each patient is provided in the Supplemental that eight (17%) donors were BKV-seronegative or equivocal, Figure 2). Rising NAb titers coincided with falling viral load in 24 (52%) recipients harbored high titers of NAbs against the urine for 19 of 24 patients who were viruric, 11 of 15 who were BKV genotype for which the donor was neutralization sero- viremic, and 11 of 13 patients with BKVAN; for 9 of 15 patients positive, whereas 14 (31%) recipients showed low NAb titers who were viremic and 9 of 13 patients with BKVAN, a falling (Supplemental Figure 3). viral load was also observed in blood (Supplemental Figure 2). The analysis of 23 matched sera from donor/BKV DNA– These kinetics could not be assessed for patients 92 and 132 positive recipient pairs at day 0 showed that one donor was (missing time points due to sample unavailability or death). seronegative. Seven (31%) BKV DNA–positive recipients har- Joint models showed that at any time, patients with high NAb bored high NAb titers against the BKV genotype for which the titers against the replicating strain were at a lower risk of BKV donor was neutralization seropositive. None of these recipi- viremia (hazard ratio [HR], 0.44; 95% confidence interval ents progressed to BKVAN and only three became viremic [95% CI], 0.26 to 0.73; P=0.002; Figure 3C, Supplemental with a noticeably short duration of viremia (37, 62, and Material). Each 1 log10 increase in NAb titer decreased the 82 days). Fifteen (65%) recipients showed low neutralizing risk of developing viremia by 56%. Recipients displaying no titers against the BKV genotype for which the donor was neu- or low NAb titer against the genotype for which their donor tralization seropositive (Supplemental Figure 3). Moreover, was seropositive were at a significantly higher risk of develop- when considering the first 10 months post-transplantation, ing viremia (HR, 2.27; 95% CI, 1.06 to 4.88; P=0.04; Figure 19 of 20 (95%) of the pairs showed a concordance between 3C). Surprisingly, the joint model analysis of urine BKV DNA the donor’s genotype-specific neutralization profile and the load at the onset of viruria or at any time before viremia onset genotype found replicating in the recipient; one recipient failed to show such a strong relationship, with an HR per log10 whose donor was BKV-seronegative developed BKV viruria at

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recipients than those for donors of BKV DNA–negative recipients (5.07 versus 3.96, P,0.001). However, donor NAb titers were similar for the three BKV DNA–positive groups (viruric, viremic, and BKVAN) (P=0.45). Using two-fold crossvalidation, we determined a NAb titer value of 4 log10 IC50 as the optimal cutoff for predicting the transition from a negative to a viruric, viremic, or BKVAN state with a C-index of 0.603, 0.635, and 0.658 for viruria, viremia, and BKVAN occurrence, respectively (Figure 4A). Indeed, a NAb titer ,4log10 IC50 on the day of transplantation was signiﬁcantly associated with BKV replication after transplantation (HR, 1.88; 95% CI, 1.06 to 3.45; P=0.03).Therewasatrendtowardahigher probability of developing viremia for patients harboring low NAb titers before transplantation (HR, 1.37; 95% CI, 0.94 to 1.96; P,0.10; Figure 4B). Eleven patients were BKV viremic (with or without BKVAN) despite displaying a baseline NAb titer .4log10.However,nineof them showed decreasing NAb titers (,4log10 for ﬁve patients) between M1 and M3 after transplantation, whereas two patients—one of whom received rituximab—showed an increase in NAb titers during follow-up without decreasing viral load, suggesting a possible immune escape mechanism.

DISCUSSION

In this study involving 168 kidney transplant recipients prospectively followed over 24 months and 69 donors, for the first time, we demonstrate that (1) recipients whodevelopdelayedandweakNAbre- sponses are at a significantly higher risk of developing BKV viremia and BKVAN; (2) on the basis of the neutralization profile, genotype mismatch between the recipients’ and donors’ strains signifi- Figure 2. BK virus (BKV) genotype–specific seroconversion after transplantation. Genotype cantly increases the risk of developing specificity was determined for each patient on the basis of NAb titers. Percentages of pa- viremia; and (3) the cutoff NAb titer value tients who were (A) BKV DNA–negative and (B) BKV DNA–positive displaying NAbs against of 4 log10 IC50 against the donor’s strain is a genotype I, II, IV, or multiple genotypes at the time of transplantation and 24 months post- robust marker that allows patient stratifica- transplantation are shown. (C) NAb titers against the replicative strain and the other geno- tion into lower and higher BKV disease risk types are depicted at month 24 for patients who were BKV DNA–positive. groups at the time of transplantation. Alto- gether, these results indicate that the 102 days post-transplantation. The three remaining recipients amount and kinetics of genotype-specific NAb titers influence developed delayed viruria at 292, 369, and 556 days after trans- BKV disease severity after transplantation. Although the BKV- plantation, suggesting an exogenous BKV reinfection. Interest- specific T cell immune response has been thoroughly investi- ingly, NAb titers were higher for donors of BKV DNA–positive gated and appears essential in controlling BKV replication,14–16

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Figure 3. A delayed and weak NAb response is associated with severe BK virus (BKV) infection outcome. (A) The evolution of NAb titers directed against the replicating strain in patients who were viruric, viremic, and those with BKVAN. NAb titer distribution is depicted in colored bars and mean NAb titer values (log10 IC50) for each group are represented by colored squares. (B) The detailed evolution of

330 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 326–334, 2018 www.jasn.org CLINICAL RESEARCH this is the first study that reveals a key role of the anti-BKV who fail to rapidly mount a NAb response against new donor- antibody–mediated neutralizing response in protecting against derived BKV genotypes, the risk of viremia is higher. Interest- BKV infection and associated diseases. ingly, analysis of donor/recipient HLA I mismatches showed Analysis of the NAb titers at the time of transplantation no difference between patients who were BKV DNA-negative, showed a similar BKV genotype distribution in BKV DNA– viruric, viremic, and those with BKVAN (Supplemental Table negative and BKV DNA–positive recipients and donors. It 1). One can assume that both cellular and humoral immune should be noted that genotype II was more prevalent than responses protect against BKV infection. However, during genotype IV, whereas only 2% of viruses were genotype II by transplantation, when a functional cellular reaction is im- sequencing samples of DNA-positive BKV recipients. Similar paired by immunosuppression, the neutralizing humoral re- findings were reported by Pastrana et al.,17 where genotype II sponse may gain importance. seroprevalence was about 20-fold higher than prior PCR- In our cohort, we observed a BKVAN incidence of 8% at based prevalence. This cannot be explained by PCR assay 2 years despite tapering of immunosuppressive therapy accord- performance because our PCR assay is able to detect and ad- ing to international guidelines, suggesting incomplete success equately quantify all BKV genotypes.18,19 On the other hand, of this pre-emptive intervention due to its delayed nature.8 this result raises the hypothesis that BKV genotype II may be Here, we demonstrate that patients displaying titers of NAbs more easily cleared compared with genotype I and IV. Further directed against the donor strain lower than the cutoff value investigations are needed to address this question. 4log10 IC50 before transplantation have a significantly in- In patients positive for BKV DNA after transplantation, we creased risk of BKV replication compared with patients har- observed a dramatic change in BKV genotype specificity of boring higher anti-BKV NAb titers. In a retrospective study, NAbs over time with an increase of NAb titers directed specif- Abend et al.22 reported that donor neutralizing serostatus cor- ically against the replicating strain but also against the other relates with incidence of post-transplant BKV viremia. In our genotypes. Similar findings were reported in mice after mono- cohort, donors whose recipients became BKV DNA–positive valent prime-boost vaccination with VLPs from individual exhibited elevated NAb titers. However, donor titers were sim- BKV subtypes that led not only to a significant increase of ilar for viruric, viremic, and BKVAN groups, suggesting that cognate neutralizing responses, but also to the development BKV disease outcome after reactivation depends on recipient of crossneutralizing NAbs.17 The possible explanation could NAb kinetics after transplantation, rather than donor character- be that high BKV replication stimulates a broader range of istics. A limitation of our study is the small number of donor/ existing memory B cells able to neutralize different BKV ge- recipient pairs investigated. Nevertheless, our findings point to a notypes and which become antibody-secreting plasma cells as key role of recipients’ NAbs specific to the donor strain and reported for other viruses.20 ThechangeofBKVgenotype demonstrate that it could be possible for patients to be further specificity over time suggests an occurrence of a de novo assigned to lower and higher BKV disease risk groups before (mainly of donor’sorigin)21 BKV infection after transplanta- transplantation. This early stratification could be useful for trans- tion. These data also confirm that patients under T cell plant physicians, potentially allowing adaptation of the immuno- immunosuppression can still develop efficient humoral re- suppressive strategy and optimal patient monitoring on the basis sponses.17 However, the kinetics of NAb responses differed of individual BKV infection risks. Furthermore, in patients who among individuals, because patients developing nephropathy develop BKV infection, monitoring of NAb titers directed against had a slower increase in BKV genotype–specific NAb titers. the replicating strain could serve as a valuable disease progression Fifty four percent of patients with BKVAN displayed a geno- marker in conjunction with viral DNA load measurement and type mismatch for their replicating strain (which is assumed to prompt early clinical management. Finally, our findings support be of donor origin),21 indicating that recipients had no NAbs the potential benefit of administering broadly NAbs as a preven- or low titers against the donor’s genotype, as opposed to only tive or therapeutic strategy against BKV infection. 17% of patients who were viruric. This finding suggests a model in which for patients displaying past immunity against a BKV genotype similar to the one found in their CONCISE METHODS matched donor, BKV replication in the graft may act as a “booster vaccination” eliciting a strong neutralizing response. Study Design Thus, NAbs appear to efficiently control BKV replication in The study was approved by the institutional review boards of the the graft and prevent transition to a viremic state. In patients Strasbourg University Hospitals (Clinical Trials.gov identifier: three representative patients of viruric, viremic, and BKVAN groups. Dotted and solid lines represent urine and blood viral loads, respectively. NAb titers are depicted by gray areas. (C) The association of NAb titers, donor/recipient genotype mismatch, andurine BKV DNA loads with the risk of transition from viruria to viremia state. Continuous or joint models were used. HR (95% CI) and P values were calculated using two-fold crossvalidation after section of the optimal cutoff at 4 log10 IC50 NAb titer. (D) Spearman’s rank correlation between NAb titers and mycophenolic acid (MMF) area under the concentration time curve (AUC), alone or associated to cyclosporine or tacrolimus. Spearman’scoefficient and associated P value are indicated.

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Figure 4. Neutralizing titers before transplantation predict the development and outcome of BK virus (BKV) infection after transplantation. Kaplan–Meier curves represent (A) BKV viruria and (B) viremia cumulative incidence according to NAb titer (log10 IC50)atthe time of transplantation (day 0 NAbs). Black curves indicate NAb titers #4log10 IC50 and green curves indicate NAb titers .4log10 IC50. Numbers at risk are indicated at each timepoint. Cumulative incidence values at month 24 are indicated.

NCT02826811). All of the subjects enrolled in this study completed a until 2 years after transplantation. The 95% limit of detection of the 19 written informed consent. No commercial sponsor was involved in PCR assay was 2.4 log10 copies per milliliter. In case of a BKV load the study. .8log10 copies per milliliter in urine, mycophenolate mofetil was re-

duced by 50%. Detection of BKV viremia at $4log10 copies per milli- Study Population liter prompted adjustment of the immunosuppressive regimen with a Adults ($18 years of age) who underwent kidney transplantation in 50% reduction of calcineurin inhibitors, a 50% reduction or cessation of Strasbourg University Hospitals between July 2012 and July 2014 mycophenolate mofetil, and introduction of leflunomide. were enrolled in this study. Serum samples from 69 matched donors Allograft biopsies were performed systematically at month 3 since were available for the study. The following two exclusion criteria were first January 2013 (141 recipients) and during 2 years of follow-up in used: (1) retransplantation due to BKVAN, and (2) no blood sample case of graft dysfunction, DSA occurrence, or sustained BKV viremia available at transplantation. On the basis of these criteria, 168 kidney (.4log10 copies per milliliter in two subsequent blood specimens). transplant recipients were included and 12 were excluded. Kidney biopsy specimens were graded according to the severity of viral cytopathic changes, inflammatory infiltrates, tubular atrophy, Immunosuppression and Management of BKV and fibrosis.23 BKVAN was diagnosed by immunohistochemistry Infection with crossreacting antibodies against the large Tantigen of the related Before transplantation, patients with preformed DSA or those un- simian polyomavirus (SV40 antibody, Calbiochem, San Diego, CA). dergoing ABO-incompatible transplantation were desensitized using BKV genotyping was performed for all BKV DNA–positive recipients rituximab and plasmapheresis. After transplantation, immuno- at different time points as previously described.19 the basis of the results suppression consisted of induction therapy with either basiliximab of BKV DNA loads in urine and blood, patients were classified as negative or thymoglobulins, calcineurin inhibitors (CNI, cyclosporine, or (no detectable BKV replication during follow-up), viruric (positive viruria tacrolimus), mycophenolate mofetil, and steroids. Steroids were in at least two consecutive samplings and negative viremia), viremic (pos- initially given at a dose of 1 mg/kg perday and were then progressively itive viruria and positive viremia), or biopsy-confirmed BKVAN.24 tapered off during the first 4 months post-transplant (for details refer to Supplemental Material). Neutralization Assay BKV replication was monitored using BKV quantitative real-time NAb titers were measured at the following time points: time of trans- PCR (BK Virus R-Gene kit, bioMérieux, Marcy l’Etoile, France)19 in plantation (day 0); months 1, 3, 6, 12, and 24; onset of viruria and urine and blood samples harvested at the time of transplantation viremia; 1 month postviruria and postviremia; and viremia clearance. (day 0), monthly for the first 6 months, and then every 3 months Neutralization assays were performed using a BKV pseudovirion

332 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 326–334, 2018 www.jasn.org CLINICAL RESEARCH system expressingthe capsid proteins of BKVgenotypes I, II, or IVas 293TT cells. We thank Rachel Freitag for her excellent technical previously described17,25 (and Supplemental Appendix). This assay assistance. enables the quantiﬁcation of antibody titers that functionally neu- This work was supported by grants from the Hôpitaux Universitaires tralize the infectivity of the different BKV genotypes, each of which de Strasbourg (Recherche Non-Interventionnelle [RNI] 2016 – N° 6368), is known to be a distinct neutralization serotype.17 BKV genotype III is the Agence Nationale de la Recherche (ANR), Laboratoire d’Excellence not represented in our study because none of our patients were in- TRANSPLANTEX (ANR-11-LABX-0070_TRANSPLANTEX), and fected by BKV genotype III (genotype III represents ,1% of BKV Institut National de la Santé et de la Recherche Médicale (UMR_S isolates in Europe and several studies suggest that BKV genotype II 1109). and III may belong to the same genotype).18,26 The neutralization titer This work was presented in abstract form on September 15, 2016 at was deﬁned as the sample dilution that yielded 50% inhibition the European Society for Clinical Virology in Lisbon, Portugal and on of pseudovirion infectivity (IC50) and was expressed as the log10 of December 8, 2016 at the Société Francophone de Transplantation in the IC50. Sera were considered non-neutralizing if the 1:100 dilution Liege, Belgium.

(2.0 log10) did not mediate at least a 50% luminometric signal reduction relative to the control condition without serum or with negative control (i.e., 50% neutralization of the reporter vector). A neutralization DISCLOSURES titer of 2.5 log10 IC50 was set as the threshold of antibody-mediated None. neutralization quantiﬁcation.

Statistical Analyses REFERENCES The distributions of continuous data were compared using nonpara- metric Mann–Whitney and Kruskal–Wallis tests when comparing 1. Hirsch HH, Knowles W, Dickenmann M, Passweg J, Klimkait T, Mihatsch different groups of patients and Wilcoxon signed-rank tests for paired MJ, Steiger J: Prospective study of polyomavirus type BK replication – comparisons. The distribution of categoric variables was compared and nephropathy in renal-transplant recipients. NEnglJMed347: 488 496, 2002 using chi-squared or Fisher exact tests. When more than two tests 2. Hirsch HH, Randhawa P; AST Infectious Diseases Community of Prac- ’ were performed for the same question, Holm s correction for multi- tice: BK polyomavirus in solid organ transplantation. Am J Transplant plicity was applied. The association between plasma concentrations 13[Suppl 4]: 179–188, 2013 of immunosuppressive drugs and NAb titers was assessed using 3. Hirsch HH, Vincenti F, Friman S, Tuncer M, Citterio F, Wiecek A, Spearman’s rank correlation. Multistate models were used to estimate Scheuermann EH, Klinger M, Russ G, Pescovitz MD, Prestele H: Poly- omavirus BK replication in de novo kidney transplant patients receiving the transition probabilities into each state of interest (viruria, vire- tacrolimus or cyclosporine: A prospective, randomized, multicenter mia, and BKVAN), and Cox proportional hazard models to assess the study. Am J Transplant 13: 136–145, 2013 association of relevant variables (NAb titers, donor/recipient geno- 4. Binet I, Nickeleit V, Hirsch HH, Prince O, Dalquen P, Gudat F, Mihatsch type mismatch, and urine BKV DNA load) with these transitions. MJ, Thiel G: Polyomavirus disease under new immunosuppressive Markov or semi-Markov formulations were adopted depending on drugs: A cause of renal graft dysfunction and graft loss. Transplantation 67: 918–922, 1999 the aims of the analyses. In addition to the association between NAb 5. Kuypers DR: Management of polyomavirus-associated nephropathy in titer and BKV DNA load measured at baseline and at each state entry, renal transplant recipients. Nat Rev Nephrol 8: 390–402, 2012 the association between the hazards of events and NAb titers or BKV 6. Seifert ME, Gunasekaran M, Horwedel TA, Daloul R, Storch GA, DNA loads measured over time was analyzed using joint models Mohanakumar T, Brennan DC: Polyomavirus reactivation and immune fi for the longitudinal process (NAb titers or BKV DNA load over responses to kidney-speci c self-antigens in transplantation. JAmSoc Nephrol 28: 1314–1325, 2017 time being alternatively considered the longitudinal processes) and 7. Hirsch HH, Babel N, Comoli P, Friman V, Ginevri F, Jardine A, fi the event process. NAb titers and BKV DNA loads were rst analyzed Lautenschlager I, Legendre C, Midtvedt K, Munoz P, Randhawa P, as continuous variables in the models, with a nonlinear effect esti- Rinaldo CH, Wieszek A: European perspective on human polyomavirus mated nonparametrically by splines. If linearity could be assumed, infection, replication and disease in solid organ transplantation. Clinl then the HR associated with the increase of one unit of the variable Microbiol Infect 20[Suppl 7]: 74–88, 2014 8. Knight RJ, Gaber LW, Patel SJ, DeVos JM, Moore LW, Gaber AO: was presented. The optimal NAb titer cutoff at baseline for the hazard Screening for BK viremia reduces but does not eliminate the risk of BK of viruria was determined using two-fold crossvalidation to limit bias nephropathy: A single-center retrospective analysis. Transplantation and over-optimism associated with cutoff selection.27 The capacity of 95: 949–954, 2013 the different predictors to discriminate between patients developing 9. Wright AJ, Gill JS: Strategies to prevent BK virus infection in kidney – the different outcomes was assessed using the concordance index.28 transplant recipients. Curr Opin Infect Dis 29: 353 358, 2016 10. Sawinski D, Forde KA, Trofe-Clark J, Patel P, Olivera B, Goral S, Bloom All analyses were performed using the R statistical software version RD: Persistent BK viremia does not increase intermediate-term graft 3.2.0 (The R Foundation for Statistical Computing, Vienna, Austria). loss but is associated with de novo donor-specificantibodies.JAmSoc Nephrol 26: 966–975, 2015 fi ACKNOWLEDGMENTS 11. Fa -Kremer S, Fofana I, Soulier E, Carolla P, Meuleman P, Leroux-Roels G, Patel AH, Cosset FL, Pessaux P, Doffoël M, Wolf P, Stoll-Keller F, Baumert TF: Viral entry and escape from antibody-mediated neutrali- We thank Christopher B. Buck for expression plasmids for VP1, VP2, zation influence hepatitis C virus reinfection in liver transplantation. J and VP3 and the National Cancer Institute Tumor Repository for Exp Med 207: 2019–2031, 2010

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334 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 326–334, 2018 Supplementary appendix

Management of immunosuppressive treatment in kidney transplant patients: ...... 2 Supplemental experimental procedure ...... 2 BKV pseudovirion production and infection...... 2 BKV pseudovirion neutralization...... 3 Figure S1...... 5 Figure S2 : ...... 9 Figure S3...... 10 Figure S4...... 11 Table S1: ...... 12 Table S2...... 13 REFERENCES ...... 13

Management of immunosuppressive treatment in kidney transplant patients:

In immunologically high-risk patients or patients with a history of acute rejection, steroids were continued at a dose of 0.1 mg/kg/day. Target trough levels of tacrolimus were 10-12 ng/ml in the first three months, 8-10 ng/ml from four to six months, and 6-8 ng/ml thereafter. Target trough levels of cyclosporine were 150–200 ng/ml in the first six months, 125–150 ng/ml from six to 12 months, and

75–125 ng/ml thereafter. The target for the mycophenolic acid area under the concentration time curve

(AUC) from 0 to 12 hours at month 1 and month 3 was 30-60 mg.h/l. In cases of acute cellular rejection, steroid pulses were administered for three days, followed by oral steroids at a dose of 1 mg/kg/day, in addition to a switch to tacrolimus in patients treated with cyclosporine.

Supplemental experimental procedure

Cells and reagents.

293TT cells (provided by the National Cancer Institute Tumor repository, Frederick, Maryland) were cultured as previously described.1 VP1 expression constructs used in this work were previously reported

1,2. Plasmids encoding synthetic codon-modified VP1, VP2 and VP3 capsid proteins of BK virus (BKV) were provided by Christopher B. Buck (National Cancer Institute, National Institute of Health, Bethesda,

Maryland). The Gaussia luciferase (GLuc) reporter plasmid phGluc was constructed by transferring an

Ecl136II-XbaI fragment of pCMV-Gluc 2 (purchased from New England BioLabs, Evry, France) into ph2m 3 cut with SnaBI and NheI.

BKV pseudovirion production and infection.

BKV pseudovirions were produced as previously described.1 Briefly, capsid VP1, VP2 and VP3 protein expression plasmids, along with reporter plasmid phGluc, were co-transfected into 293TT cells. Two days after transfection, the cells were lysed and pseudovirions maturated overnight using 0.1% of

Ambion® RNase Cocktail™ (ThermoFischer Scientific, Illkirch-Graffenstaden, France). The pseudovirions were harvested and purified by ultracentrifugation through a iodixanol step gradient

(Optiprep, Sigma-Aldrich, Saint-Quentin Fallavier, France). To minimize any variation, a single pseudovirion stock was produced for each genotype, aliquoted and used for all the experiments. To analyze BKV pseudovirion infectivity, pseudovirions were added to 293TT cells and incubated for 72h at 37°C. BKV pseudovirion infectivity was determined by analysis of luciferase reporter gene expression as described previously.1 The detection limit for positive luciferase reporter protein expression was 103 RLU/assay corresponding to the mean ± 3 SD of background levels, i.e., luciferase activity of the supernatant of naïve noninfected cells.

BKV pseudovirion neutralization.

For the study of antibody-mediated neutralization, BKV pseudovirions were mixed with serially diluted patient serum, serially diluted positive control (consisting of a pool of >100 anti-BKV positive sera), or negative control (consisting of an anti-BKV-negative serum), and preincubated for 1 h at 4°C and added to 293TT cells for 72 h at 37°C.1 BKV pseudovirion neutralization was determined by analysis of luciferase reporter gene expression as described previously.1 The neutralization titer was defined as the sample dilution that yielded 50% inhibition of pseudovirion infectivity (IC50) and was expressed as the log10 of the IC50. Sera were considered non-neutralizing if the 1:100 dilution (2.0 log10) did not mediate at least a 50% luminometric signal reduction relative to the control condition without serum or with negative control (i.e., 50% neutralization of the reporter vector). A neutralization titer of 2.5 log10 IC50 was set as the threshold of antibody-mediated neutralization quantification.

The results between 2.00 and 2.50 log10 were classified as "equivocal" since the calculation of the extrapolation of the sigmoidal curve of the IC50 depends strongly on the first dilution well, while the calculation of the higher titers takes into account two to eight dilution measurements, ensuring higher robustness.

To ensure the reproducibility of our assay, a positive control was introduced into each experiment and the titer was followed over time with a Levey-Jennings plot. For example, for genotype I, the mean value of the positive control NAb titer was 4.25 with a standard deviation of 0.19 (ie, 1.5 times the change). The robustness of the analysis was evaluated by a double test of the IC50 for 92 samples. The

3 median difference between the first and second measurements was 0.28 log10 (95% confidence interval,

0.19 to 0.37 log10), that is, 1.9 times the change.

This neutralizing assay is able to fully discriminate between the different BKV genotypes. Although

BKV genotypes have significant homology (Figure S4), by using serological analysis of human sera and sera from mice given virus-like particle (VLP)-based BKV vaccines, Pastrana et al. demonstrated that the different BKV genotypes are fully distinct serotypes 1,2, the difference being governed by few residues in the VP1 sequence adjacent to the cellular glycan receptor-binding site on the virion surface

Figure S1. BK virus (BKV) genotype-specific serology of kidney-donors. Genotype specificity was determined for each donor based on neutralizing antibody (NAb) titers. Percentages of donors displaying

NAbs against genotype I, II, IV or multiple genotypes are shown.

b denotes neutralizing antibodies and

and solid dotted by depicted are blood

. Viral loads in urine and in urine loads . Viral

positive patients positive -

V DNA V . BKVAN diagnosis is indicated by an arrow. NA

titers are shown in grey areas

associated nephropathy. nephropathy. associated - Viral load and NAb titer evolution in BK in titer evolution NAb and load Viral

2 S AN, BKV re , respectively. NAb Figu lines BKV

Figure S3. Donor/recipient serostatus in BKV DNA-negative and BKV DNA-positive patients. Pie charts show the percentages of seronegative donor and recipients with low or high neutralizing antibody (NAb) titers against the donor strain for BKV DNA-negative patients (Panel A) and BKV DNA-positive patients (Panel B).

Figure S4. Amino acid sequences of the major capsid protein VP1 for BK virus (BKV) genotype

I, II and IV. Deduced amino acid sequences of VP1 are depicted for the 3 BKV genotypes. Dots indicate amino acid identity and residues indicate amino acid changes between the BKV genotypes. The box indicates the BC loop of VP1.

Table S1: Patient characteristics in BKV DNA-negative, viruric, viremic and BKVAN groups.

BKV DNA- viruric viremic BKVAN Variable negative P values (n=24) (n=15) (n=13) (n=116)

Age (median) 55.8 53.1 49.3 62.8 0.05

Male (%) 61.9 52.4 80.0 53.8 0.36

1st graft (%) 80.9 61.9 53.3 76.9 0.27

Living donor (%) 13.8 33.3 6.7 7.7 0.06

HLA I mismatches (median) 2.5 2.1 2.6 3.1 0.27

Cold ischemia time (minutes, 834 647 927 838 0.16 median)

Thymoglobulin (%) 52.4 47.6 66.7 38.4 0.49

Tacrolimus (%) 52.4 57.1 46.7 61.5 0.87

Rituximab (%) 0.9 0.0 6.7 23.1 < 0.001

Kruskall-Wallis test p-values are indicated. BKV denotes BK virus and BKVAN BK virus-associated nephropathy.

Table S2. Landmark analysis at 1 month after transplantation

Viremia risk * Variables Hazard ratio Concordance index

1-month NAb titer 0.44 (0.26 – 0.75) 0.708 (0.574- 0.842)

1-month viruria 1.21 (0.84 – 1.72) 0.600 (0.463 – 0.738)

*To assess the yield of repeated measurements of NAb titers during follow-up on the prediction of viremia, a landmark analysis at one month was conducted. The prognostic value of NAb titer and viral load at one month from viruria was therefore assessed in the subset of patients who developed viruria and were still followed and viremia-free after one month4. Using a landmark analysis at 1 month, we can show that the 1-month NAb titers are associated with an increased viremia risk after 1 month of viruria, with a HR of 0.44 (0.26 to 0.75).The same analyses for viral load failed to show such a strong relationship, with a HR per log10 increase of 1.21 (0.84 to 1.72) in the joint model, and 1.17 (0.87 to 1.58) for viral load at 1 month in the landmark analysis. The c-index for the 1-month NAb titer for predicting viremia was 0.708 (0.574 to 0.842), indicating good discrimination, markedly better than for viral load (0.600, 95%CI 0.463 to 0.738), although the difference was not formally significant (p=0.23). Altogether, these data suggest that in comparison to viral load measurements, repeated measures of NAb titers significantly improve prediction and allow better patient stratification.

REFERENCES

1. Pastrana DV, Brennan DC, Cuburu N, et al. Neutralization serotyping of BK polyomavirus infection in kidney transplant recipients. PLoS Pathog 2012;8:e1002650.

2. Pastrana DV, Ray U, Magaldi TG, Schowalter RM, Çuburu N, Buck CB. BK polyomavirus genotypes represent distinct serotypes with distinct entry tropism. J Virol 2013;87:10105-13.

3. Harrell FE, Jr., Lee KL, Mark DB. Multivariable prognostic models: issues in developing models, evaluating assumptions and adequacy, and measuring and reducing errors. Statistics in medicine

1996;15:361-87.

SIGNIFICANCE STATEMENT

BKvirus-associated nephropathy(BKVAN) isoneof the major causes of graft dysfunction and loss in kidney transplantation. The management of BKVAN is challenging, partly due to the lack of predictive markers. This manuscript describes the discovery of BKV genotype-specific neutralizing antibody titers as a new predictive marker for BKVAN. We demonstrate that recipients who develop weak antibody neutralizing responses are at a significantly higher risk of developing BKVAN. In addition, we identified a cutoff value that allows patient stratification into lower and higher BKV disease risk groups at the time of transplantation. Our findings offer a useful tool for clinicians to improve kidney transplant management and pave the way for the development of new preventive or therapeutic strategies.