Adrian Egli

Prevalence of Polyomavirus BK and JC Infection and Replication in 400 Healthy Blood Donors

BACKGROUND The replication of BK virus (BKV) and JC virus (JCV) is linked to polyomavirus-associated nephropathy, hemorrhagic cystitis, and multifocal leukoencephalopathy in immunodeficient patients, but the behavior of these viruses in immunocompetent individuals has hardly been characterized. METHODS We used EIA to study samples obtained from 400 healthy blood donors aged 20-59 years for BKV- and JCV-specific antibodies against virus-like particles. We also studied BKV and JCV loads in plasma and urine among these individuals by use of real-time polymerase chain reaction. RESULTS IgG seroprevalence was 82% (328 of 400 donors) for BKV and 58% (231 of400) for JCV. As age increased (age groups were divided by decade), the seroprevalence of BKV decreased from 87% (87 of 100) in the youngest group (aged 20-29 years) to 71% (71 of 100) in the oldest group (aged 50-59 years) (P = .006), whereas the seroprevalence of JCV increased from 50% (50 of 100) in the youngest group to 68% (68 of 100) in the oldest group (P = .06). Asymptomatic urinary shedding of BKV and JCV was observed in 28 (7%) and 75 (19%) of 400 subjects, respectively, with median viral loads of 3.51 and 4.64 log copies/mL, respectively (P < .001). Unlike urinary BKV loads, urinary JCV loads were positively correlated with IgG levels. The shedding of JCV was more commonly observed among individuals who were seropositive only for JCV, compared with individuals who were seropositive for both BKV and JCV, suggesting limited cross-protection from BKV immunity. Noncoding control regions were of archetype architecture in all cases, except for 1 rearranged JCV variant. Neither BKV nor JCV DNA was detected in plasma. CONCLUSIONS Our study provides important data about polyomavirus infection and replication in healthy, immunocompetent individuals. These data indicate significant differences between BKV and JCV with respect to virus-host interaction and epidemiology.

Polyomavirus BK-Specific Cellular Immune Response to VP1 and Large T-Antigen in Kidney Transplant Recipients

Polyomavirus BK (BKV) is the primary cause of polyomavirus‐associated nephropathy (PVAN) in kidney transplant (KT) recipients. Using ELISpot assays, we compared the frequency of interferon‐γ (IFN‐γ) secreting peripheral blood mononuclear cells (PBMC) after stimulation with overlapping peptide pools covering BKV large T‐antigen (LT) and VP1 capsid proteins (VP1). In 10 healthy donors, LT and VP1 responses were low with median 24 (range 15–95) and 25 (7–113) spot‐forming units/106 PBMC (SFU), respectively. In 42 KT patients with current or recent plasma BKV loads, median LT and VP1 responses of 29 (0–524) and 114 (0–1432) SFU were detected, respectively. In KT patients with decreasing or past plasma BKV loads, significantly higher median BKV‐specific IFN‐γ responses were detected compared to KT patients with increasing or persisting BKV loads [LT: 78 (8–524) vs. 22 (0–120) SFU, p = 0.003; VP1: 285 (45–1432) vs. 53 (0–423) SFU, p = 0.001, respectively]. VP1‐specific IFN‐γ responses were higher and more likely to involve CD4+ T cells, while CD8+ T cells were more frequently directed against LT. Stimulation with JCV‐specific VP1 and LT peptides indicated only low‐level cross‐recognition. The data suggest that control of BKV replication is correlated with differentiated expansion of BKV‐specific cellular immune responses.

Polyomavirus BK with rearranged noncoding control region emerge in vivo in renal transplant patients and increase viral replication and cytopathology

Immunosuppression is required for BK viremia and polyomavirus BK–associated nephropathy (PVAN) in kidney transplants (KTs), but the role of viral determinants is unclear. We examined BKV noncoding control regions (NCCR), which coordinate viral gene expression and replication. In 286 day–matched plasma and urine samples from 129 KT patients with BKV viremia, including 70 with PVAN, the majority of viruses contained archetypal (ww-) NCCRs. However, rearranged (rr-) NCCRs were more frequent in plasma than in urine samples (22 vs. 4%; P < 0.001), and were associated with 20-fold higher plasma BKV loads (2.0 × 104/ml vs. 4.4 × 105/ml; P < 0.001). Emergence of rr-NCCR in plasma correlated with duration and peak BKV load (R2 = 0.64; P < 0.001). This was confirmed in a prospective cohort of 733 plasma samples from 227 patients. For 39 PVAN patients with available biopsies, rr-NCCRs were associated with more extensive viral replication and inflammation. Cloning of 10 rr-NCCRs revealed diverse duplications or deletions in different NCCR subregions, but all were sufficient to increase early gene expression, replication capacity, and cytopathology of recombinant BKV in vitro. Thus, rr-NCCR BKV emergence in plasma is linked to increased replication capacity and disease in KTs.

Inhibition of polyomavirus BK-specific T-Cell responses by immunosuppressive drugs

Background. Reducing immunosuppression is the treatment of choice for polyomavirus-associated nephropathy in kidney transplant (KT) patients, but strategies and targets are uncertain. Methods. Using interferon-&ggr; ELISpot assays, we investigated immunosuppressive drug levels and polyomavirus BK (BKV) large T-antigen-specific T-cell responses in KT patients in vivo and in healthy donors after titrating immunosuppression in vitro. Results. In KT patients, BKV-specific T-cell responses were inversely correlated with tacrolimus trough levels (R2=0.28, P<0.002), but not with mycophenolate levels, prednisone, or overall immunosuppressive dosing. In vitro tacrolimus concentrations above 6 ng/mL inhibited BKV- and cytomegalovirus-specific T-cells more than 50%, whereas less than 30% inhibition was observed below 3 ng/mL. Inhibition by cyclosporine A was more than 50% at concentrations of 1920 ng/mL and less than 30% below 960 ng/mL, corresponding to clinical C0 trough levels of 200 and 100 ng/mL, respectively. However, mycophenolate up to 8 &mgr;g/mL, leflunomide 50 &mgr;g/mL, or sirolimus concentrations 64 ng/mL did not inhibit BKV-specific interferon-&ggr; production, but antigen-dependent T-cell expansion. Conclusions. Calcineurin-inhibitor concentrations are critical for BKV-specific T-cell activation. Reducing calcineurin inhibitors should be considered as first step, whereas conversion to mTOR inhibitors may be an attractive alternative or second step that should be validated in clinical BKV intervention trials.

Cytomegalovirus-specific T-cell responses and viral replication in kidney transplant recipients

BackgroundCytomegalovirus (CMV) seronegative recipients (R-) of kidney transplants (KT) from seropositive donors (D+) are at higher risk for CMV replication and ganciclovir(GCV)-resistance than CMV R(+). We hypothesized that low CMV-specific T-cell responses are associated with increased risk of CMV replication in R(+)-patients with D(+) or D(-) donors.MethodsWe prospectively evaluated 73 consecutive KT-patients [48 R(+), 25 D(+)R(-)] undergoing routine testing for CMV replication as part of a preemptive strategy. We compared CMV-specific interferon-γ (IFN-γ) responses of CD4+CD3+ lymphocytes in peripheral blood mononuclear cells (PBMC) using three different antigen preparation (CMV-lysate, pp72- and pp65-overlapping peptide pools) using intracellular cytokine staining and flow cytometry.ResultsMedian CD4+ and CD8+T-cell responses to CMV-lysate, pp72- and pp65-overlapping peptide pools were lower in D(+)R(-) than in R(+)patients or in non-immunosuppressed donors. Comparing subpopulations we found that CMV-lysate favored CD4+- over CD8+-responses, whereas the reverse was observed for pp72, while pp65-CD4+- and -CD8+-responses were similar. Concurrent CMV replication in R(+)-patients was associated with significantly lower T-cell responses (pp65 median CD4+ 0.00% vs. 0.03%, p = 0.001; CD8+ 0.01% vs. 0.03%; p = 0.033). Receiver operated curve analysis associated CMV-pp65 CD4+ responses of > 0.03% in R(+)-patients with absence of concurrent (p = 0.003) and future CMV replication in the following 8 weeks (p = 0.036). GCV-resistant CMV replication occurred in 3 R(+)-patients (6.3%) with pp65- CD4+ frequencies < 0.03% (p = 0.041).ConclusionThe data suggest that pp65-specific CD4+ T-cells might be useful to identify R(+)-patients at increased risk of CMV replication. Provided further corroborating evidence, CMV-pp65 CD4+ responses above 0.03% in PBMCs of KT patients under stable immunosuppression are associated with lower risk of concurrent and future CMV replication during the following 8 weeks.

State-of-the-Art Monitoring of Cytomegalovirus-Specific Cell-Mediated Immunity After Organ Transplant: A Primer for the Clinician

Cytomegalovirus (CMV) is one of the most common infections after solid organ transplantation. Improved assays to predict viral replication and disease would help refine current preventive strategies. Monitoring of CMV-specific T-cell responses may help guide clinical decision making. Several techniques are now available to quantify CMV-specific T-cell responses, including flow cytometry, enzyme-linked immunosorbent spot assay, and enzyme-linked immunosorbent assay. Standardization and validation of these assays have the potential to significantly change the monitoring and treatment of CMV and further personalize CMV prevention strategies. In this review, we discuss the measurement of CMV-specific T-cell responses and their clinical impact on the management of CMV after organ transplantation.

Randomized Controlled Trial of High-Dose Intradermal Versus Standard-Dose Intramuscular Influenza Vaccine in Organ Transplant Recipients

The immunogenicity of standard intramuscular (IM) influenza vaccine is suboptimal in transplant recipients. Also, recent studies suggest that alloantibody may be upregulated due to vaccination. We evaluated a novel high‐dose intradermal (ID) vaccine strategy. In conjunction, we assessed alloimmunity. Transplant recipients were randomized to receive IM or high‐dose ID vaccine. Strain‐specific serology and HLA alloantibody production was determined pre‐ and postimmunization. In 212 evaluable patients (105 IM, 107 ID), seroprotection to H1N1, H3N2 and B strains was 70.5%, 63.8% and 52.4% in the IM group, and 71.0%, 70.1%, 63.6% in the ID group (p = ns). Seroconversion to ≥1 antigen was 46.7% and 51.4% in the IM and ID groups respectively (p = 0.49). Response was more likely in those ≥6 months posttransplant (53.2% vs. 19.2%; p = 0.001). Use of mycophenolate mofetil was inversely associated with vaccine response in a dose‐dependent manner (p < 0.001). Certain organ subgroups had higher response rates for influenza B in the ID vaccine group. Differences in anti‐HLA antibody production were detected in only 3/212(1.4%) patients with no clinical consequences. High‐dose intradermal vaccine is an alternative to standard vaccine and has potential enhanced immunogenicity in certain subgroups. In this large cohort, we also show that seasonal influenza does not result in significant alloantibody production.

IL-28B is a Key Regulator of B- and T-Cell Vaccine Responses against Influenza

Influenza is a major cause of morbidity and mortality in immunosuppressed persons, and vaccination often confers insufficient protection. IL-28B, a member of the interferon (IFN)-λ family, has variable expression due to single nucleotide polymorphisms (SNPs). While type-I IFNs are well known to modulate adaptive immunity, the impact of IL-28B on B- and T-cell vaccine responses is unclear. Here we demonstrate that the presence of the IL-28B TG/GG genotype (rs8099917, minor-allele) was associated with increased seroconversion following influenza vaccination (OR 1.99 p = 0.038). Also, influenza A (H1N1)-stimulated T- and B-cells from minor-allele carriers showed increased IL-4 production (4-fold) and HLA-DR expression, respectively. In vitro, recombinant IL-28B increased Th1-cytokines (e.g. IFN-γ), and suppressed Th2-cytokines (e.g. IL-4, IL-5, and IL-13), H1N1-stimulated B-cell proliferation (reduced 70%), and IgG-production (reduced>70%). Since IL-28B inhibited B-cell responses, we designed antagonistic peptides to block the IL-28 receptor α-subunit (IL28RA). In vitro, these peptides significantly suppressed binding of IFN-λs to IL28RA, increased H1N1-stimulated B-cell activation and IgG-production in samples from healthy volunteers (2-fold) and from transplant patients previously unresponsive to vaccination (1.4-fold). Together, these findings identify IL-28B as a key regulator of the Th1/Th2 balance during influenza vaccination. Blockade of IL28RA offers a novel strategy to augment vaccine responses.

Viral Impact on Long-term Kidney Graft Function

Acute rejection episodes are an important risk factor for the functional deterioration of solid-organ transplants. With more intense immunosuppressive protocols, the rate of acute rejection episodes has significantly declined in the last decade, but long-term graft function and graft survival are challenged by increasing viral complications. In this article, recent data on the role of adenovirus, polyomavirus BK and JC, cytomegalovirus, human herpesvirus-6 and -7, and parvovirus B19 on the long-term outcome of kidney transplantation are reviewed. An update on the pathophysiology of smoldering viral replication, associated inflammatory damage, and the presumed indirect viral effects is provided, and the implications for diagnostic tests and antiviral intervention are discussed.

Renal failure five years after lung transplantation due to polyomavirus BK-associated nephropathy.

Polyomavirus‐associated nephropathy (PyVAN) is rare in nonrenal solid organ transplantation and only limited information is available from single cases. We describe a 67‐year‐old female presenting with hypertension and progressive kidney failure due to PyVAN 60 months after lung transplantation. Plasma BK virus (BKV) loads were 4.85 log10 copies/mL at diagnosis and cleared slowly over 14 months after switching from tacrolimus, mycophenolate and prednisone to low‐dose tacrolimus, sirolimus and leflunomide, the latter being discontinued for anemia and diarrhea. BKV‐ and JC virus‐specific immunoglobulins were detectable prior to transplantation. Only BKV‐specific IgG and IgM increased during follow‐up. BKV‐specific T cells were detectable in blood following in vitro expansion, but cleared with reincreased sirolimus, yet BKV viremia remained undetectable. We identified eight other cases of PyVAN in nonrenal solid organ transplantation including lung (n = 1), heart (n = 6) and pancreas (n = 1). Overall, diagnosis was later than commonly seen in kidney transplants (median 18 months, interquartile range 10–29). Seven patients were male, five received triple immunosuppression consisting of tacrolimus, mycophenolate, prednisone. Immunosuppression was reduced in four cases and cidofovir and/or leflunomide administered in five and two cases, respectively. Renal function deteriorated in five requiring hemodialysis in four. We discuss mTOR inhibitors versus cidofovir and leflunomide as potential PyVAN rescue therapy.