Immunosuppressive strategies in renal transplantation aim to improve renal function, to prolong graft survival and to minimize the occurrence of adverse effects . Standard immunosuppressive regimens in renal transplantation generally consist of calcineurin inhibitors (CNIs) [tacrolimus or ciclosporin], mycophenolate (mycophenolate mofetil [MMF] or entericcoated mycophenolate sodium), and corticosteroids (methylprednisolone or prednisolone) . The addition of induction therapy with antilymphocyte antibodies or interleukin (IL)-2 receptor (IL-2Rα) antibodies, such as basiliximab (Simulect®; Novartis, Basel, Switzerland), to standard immunosuppressive regimens has reduced the risk of acute rejection episodes during the early post-transplant period when the risk of rejection is greatest [1, 3]. Basiliximab is a recombinant chimeric IgG1 monoclonal antibody that binds specifically to the α-subunit of the IL-2Rα (also referred to as the CD25 antigen) on activated T cells, thereby inhibiting IL-2-mediated proliferation of T lymphocytes, a critical step in the cellular immune response involved in allograft rejection . Basiliximab induction allows dose reduction of corticosteroids or CNIs, thereby minimizing the adverse effects associated with these co-administered agents. Moreover, the addition of basiliximab to a triple immunotherapy containing azathioprine or MMF resulted either in a significantly reduction in the incidence of biopsy-confirmed acute rejection episodes (40.4% and 42.5%, respectively) at 6 months when compared with placebo either in an increase of the IL-2Rα saturation period (36 vs 50 days and 36 vs 59 days, respectively) [2, 4]. Thus, current treatment guidelines recommend the use of basiliximab as part of a CNI-based regimen for the prophylaxis of acute graft rejection in renal transplantation in adults, adolescents and children [5–8].
The introduction in clinical practice of newer, more potent immunosuppressive agents has been correlated with the higher prevalence of polyomavirus-associated nephropathy (PVAN) or, more specifically, BK polyomavirus-associated nephropathy (BKVAN) in renal transplant patients, indicating a relationship between the human polyomavirus BK (BKV) reactivation and the disruption of the immune system [9–11]. BKVAN is characterized by necrosis of proximal tubules and denudation of the basement membrane as a result of BKV lytic infection in kidney epithelial cells [12, 13], but is often misdiagnosed as acute rejection or drug toxicity [14, 15]. It occurs in 1% to 10% of kidney transplant recipients, usually manifesting in the first year following transplantation and leading to graft loss in 50% of infected patients within 2-3 years of follow-up [10, 14].
The current recommendation includes screening for BKV reactivation with subsequent preventive reduction of immunosuppression with or without antiviral therapy . Among all available diagnostic methods for BKV infection, analysis of BKV DNA load in plasma has been shown to have the highest predictive value for BKVAN with BKV load in plasma ≥ 104 copies/mL for > 3 weeks .
BKV infection is ubiquitous among the human population from early childhood, with a seroprevalence in adults of more than 80% . After primary infection, BKV persists in a latent state in cells of several organs including kidney (the main site of BKV latency in healthy individuals), peripheral blood leukocytes, and maybe other sites such as the lung, eye, liver, and brain [9, 19]. BKV belongs to the Polyomaviridae family, which includes non-enveloped DNA viruses with icosahedral capsids of 45 nm diameter containing small, circular double-stranded DNA genomes of approximately 5 Kb [20, 21]. The viral genome is divided into early, late, and regulatory regions and encodes for at least six proteins, two from the early region [the large tumor antigen (TAg) and the small tumor antigen (tAg)] and four from the late region [the capsid proteins VP1, VP2, VP3 and the agnoprotein][21, 22]. VP1, which is the major capsid protein, is present on the surface of the capsid and is responsible for receptor binding to host cells. In addition, VP1 is highly immunogenic, is the target of neutralizing antibody and is required for virion assembly and hemagglutination of red blood cells. Four distinct serotypes of BKV have been previously determined by hemagglutination inhibition, named subtypes I-IV by Jin et al. . Genetic analyses of VP1 sequences have determined that the serotyping region contains a variable region of the BKV genome between nucleotides 1744 and 1812 (amino acids 61 to 83). Subtype I (further divided into four subgroups, each of which has a unique geographical distribution pattern: I/a, I/b-1, I/b-2 and I/c) is the most frequent in the normal human population worldwide (80%), subtype IV (further divided into six subgroups with their own geographical distribution pattern: IV/a-1, IV/a-2, IV/b-1, IV/b-2, IV/c-1 and IV/c-2) is prevalent in Asia and part of Europe, while subtypes II and III are infrequently detected in normal adults . Several evolutionary studies using phylogenetic analysis suggest a co-migration of BKV and the human race to explain the geographical distribution patterns of BKV subtypes and subgroups [24–27].
The regulatory region contains the origin of DNA replication (O-block, 142 base-pairs) and sequences involved in transcriptional regulation of both the early and the late genes (promoter/enhancer elements), referred to as the transcriptional control region (TCR). The TCR of the proposed archetypal BK strain WW (WW TCR) has been arbitrarily divided into four transcription factor binding sequence blocks, called P (68 base-pairs), Q (39 base-pairs), R (63 base-pairs), and S (63 base-pairs). The different BKV strains display a marked heterogeneity in their TCR due to point mutations, deletions, duplications, and rearrangements in this region . These rearrangements may play an important role in virus replication by increasing or decreasing the number or the affinities of host transcription factor binding sites [28, 29]. BKV variants with rearranged TCR have been identified in various studies including kidney transplant recipients . The BKV variants with rearranged TCR emerging as the majority species in blood of recipients with BKV-nephropathy are quite heterogeneous with insertions, deletions, and mixtures, but all seem to confer an increased early gene expression, a higher replication capacity and more pronounced cytopathology in cell culture compared to archetype WW TCR. Interestingly, the displacement of archetype TCR by rearranged TCR was not matched by a simultaneous displacement in the urine indicating that these sites represent largely independent replication compartments .
As mentioned above, although in the last decade better immunosuppression drugs, including basiliximab as induction therapy, have decreased the rates of acute rejection in kidney transplantation, they have also led to the emergence of BKVAN, that may occur early after kidney transplantation [16, 31, 32]. Therefore, the aim of our study was to prospectively investigate the BKV viral load in plasma and urine samples belonging to a cohort of 60 adult kidney transplant patients, treated with basiliximab combined with a MMF-based triple immunotherapy, during the first 6 months post-transplantation to monitor the trend of BKV viremia and viruria, focusing our attention on the difference between viral replication during the first 3 months, characterized by the immunosuppressive action of basiliximab, and the following 3 months, when the monoclonal antibody was completely removed and the maintenance therapy acted alone. We also performed sequencing analysis of all BKV positive samples to assess the presence of BKV TCR variants and sequencing analysis of the VP1 region to determine whether a particular BKV subtype/subgroup was more present in these anatomic areas.