Gang Zeng

Banff Initiative for Quality Assurance in Transplantation (BIFQUIT): Reproducibility of Polyomavirus Immunohistochemistry in Kidney Allografts

Immunohistochemistry (IHC) is the gold standard for diagnosing (positive vs. negative) polyomavirus BK (BKV) nephropathy and has the potential for disease staging based on staining intensity and quantification of infected cells. This multicenter trial evaluated the reproducibility of BKV IHC among 81 pathologists at 60 institutions. Participants stained tissue microarray slides and scored them for staining intensity and percentage of positive nuclei. Staining protocol details and evaluation scores were collected online. Slides were returned for centralized panel re‐evaluation and kappa statistics were calculated. Individual assessment of staining intensity and percentage was more reproducible than combined scoring. Inter‐institutional reproducibility was moderate for staining intensity (κ = 0.49) and percentage (κ = 0.42), fair for combined (κ = 0.25) and best for simple positive/negative scoring (κ = 0.78). Inter‐observer reproducibility was substantial for intensity (κ = 0.74), percentage (κ = 0.66), positive/negative (κ = 0.78) and moderate for combined scoring (κ = 0.43). Inter‐laboratory reproducibility was fair for intensity (κ = 0.37), percentage (κ = 0.40) and combined (κ = 0.24), but substantial for positive/negative scoring (κ = 0.67). BKV RNA copies/cell correlated with staining intensity (r = 0.56) and percentage (r = 0.62). These results indicate that BKV IHC is reproducible between observers but scoring should be simplified to a single‐feature schema. Standardization of tissue processing and staining protocols would further improve inter‐laboratory reproducibility.

Commercially Available Immunoglobulins Contain Virus Neutralizing Antibodies Against All Major Genotypes of Polyomavirus BK

Neutralizing antibodies (NAbs) form the basis of immunotherapeutic strategies against many important human viral infections. Accordingly, we studied the prevalence, titer, genotype‐specificity, and mechanism of action of anti‐polyomavirus BK (BKV) NAbs in commercially available human immune globulin (IG) preparations designed for intravenous (IV) use. Pseudovirions (PsV) of genotypes Ia, Ib2, Ic, II, III, and IV were generated by co‐transfecting a reporter plasmid encoding luciferase and expression plasmids containing synthetic codon‐modified VP1, VP2, and VP3 capsid protein genes into 293TT cells. NAbs were measured using luminometry. All IG preparations neutralized all BKV genotypes, with mean EC50 titers as high as 254 899 for genotype Ia and 6,666 for genotype IV. Neutralizing titers against genotypes II and III were higher than expected, adding to growing evidence that infections with these genotypes are more common than currently appreciated. Batch to batch variation in different lots of IG was within the limits of experimental error. Antibody mediated virus neutralizing was dose dependent, modestly enhanced by complement, genotype‐specific, and achieved without effect on viral aggregation, capsid morphology, elution, or host cell release. IG contains potent NAbs capable of neutralizing all major BKV genotypes. Clinical trials based on sound pharmacokinetic principles are needed to explore prophylactic and therapeutic applications of these anti‐viral effects, until effective small molecule inhibitors of BKV replication can be developed.

Antigen-Specificity of T Cell Infiltrates in Biopsies With T Cell-Mediated Rejection and BK Polyomavirus Viremia: Analysis by Next Generation Sequencing.

This study interrogates the antigen‐specificity of inflammatory infiltrates in renal biopsies with BK polyomavirus (BKPyV) viremia (BKPyVM) with or without allograft nephropathy (BKPyVN). Peripheral blood mononuclear cells (PBMC) from five healthy HLA‐A0101 subjects were stimulated by peptides derived from the BKPYV proteome or polymorphic regions of HLA. Next generation sequencing of the T cell–receptor complementary DNA was performed on peptide‐stimulated PBMC and 23 biopsies with T cell–mediated rejection (TCMR) or BKPyVN. Biopsies from patients with BKPyVM or BKVPyVN contained 7.7732 times more alloreactive than virus‐reactive clones. Biopsies with TCMR also contained BKPyV‐specific clones, presumably a manifestation of heterologous immunity. The mean cumulative T cell clonal frequency was 0.1378 for alloreactive clones and 0.0375 for BKPyV‐reactive clones. Samples with BKPyVN and TCMR clustered separately in dendrograms of V‐family and J‐gene utilization patterns. Dendrograms also revealed that V‐gene, J‐gene, and D‐gene usage patterns were a function of HLA type. In conclusion, biopsies with BKPyVN contain abundant allospecific clones that exceed the number of virus‐reactive clones. The T cell component of tissue injury in viral nephropathy appears to be mediated primarily by an “innocent bystander” mechanism in which the principal element is secondary T cell influx triggered by both antiviral and anti‐HLA immunity.

Severe Acute T Cell and Antibody-Mediated Rejection in Ectopic Kidney Allografts With or Without Mouse Polyomavirus Infection

Figure 1: Histologic findings in an allograft kidney from a mouse at the time of peak viremia, 7 days postinfection. There is severe interstitial mononuclear inflammation (A) with intimal arteritis (B). Diffuse C4d deposition in the peritubular capillaries (C) indicates that antibody mediated rejection is also contributing to the graft injury. No MPyV T-antigen could be documented in the renal tubules (data not shown) using an immunohistochemical staining procedure that worked successfully on a virus infected 3T3 cell line used as a positive control (D). A color version of this figure is available online. animal models of this disease. A study recently published in the journal investigated how several clinically relevant variables affect the magnitude of kidney injury in this model (1). No viral antigens were demonstrated in renal tubules.

Inhibition of large T antigen ATPase activity as a potential strategy to develop anti-polyomavirus JC drugs.

INTRODUCTION This study evaluates polyomavirus JC (JCV) large T antigen (LTA) as a potential target for drug development. LTA is a hexameric protein with a helicase activity that is powered by ATP binding and hydrolysis. The helicase and ATPase function is critical for viral replication. METHODS Recombinant JCV LTA was produced in an Escherichia coli based expression plasmid. ATPase activity was measured using the malachite green assay. A high throughput screen was completed using a brain-biased library of 75,000 drug-like compounds selected for physicochemical properties consistent with blood-brain barrier permeability. RESULTS Five compounds showed non-competitive inhibition of ATPase activity with an EC50 ⩽ 15 μM. Modest antiviral activity was demonstrated in an immunofluorescence assay for JCV VP-1 expression in COS7 cells (EC50 15, 18, 20, 27, and 52 μM respectively). The compounds also inhibited viral replication in a real time PCR assay at comparable concentrations. LD50 in the MTS96 and Cell TiterGlo assays was >100 μM for all compounds in COS7 as well as HEK293 cells. However, two compounds inhibited cell proliferation in culture with IC50 values of 43 and 34 μM respectively. Despite substantial amino acid similarity between polyomavirus JC, BK and SV40 proteins, these compounds differ from those previously reported to inhibit SV40 LTA ATPase in chemical structure as well as a non-competitive mechanism of inhibition. CONCLUSION LTA ATPase is a valid target for discovery. Additional screening and chemical optimization is needed to develop clinically useful compounds with less toxicity, which should be measured by metabolic as well as cell proliferation assays.

Validation of BKV large T-antigen ATP-binding site as a target for drug discovery

BK virus large T antigen (LTA) is a hexameric protein with a helicase activity that is powered by ATP hydrolysis. A mutant virus with Lys420Ala, Arg421Ala, and Asp504Ala mutations at the ATP binding sites showed marked reduction in viral fitness. This observation indicates that high throughput screening for ATPase inhibitors will be valid strategy to discover anti-BKV drugs. Pilot screening of 300 compounds from the Tim Tec ActiTarg K library identified a compound, STO18584, with selectivity index of 19.2.

Rejection of the Renal Allograft in the Absence of Demonstrable Antibody and Complement

BackgroundRecent literature has stressed the prominent role of antibodies in graft loss. This study was designed to assess a growing perception that T cell-mediated rejection (TCMR) is no longer clinically relevant. MethodsFive hundred forty-five renal allograft recipients over a 3-year period were screened for biopsies with: (a) TCMR including borderline change (BL), (b) negative complement protein C4 degradation fragment, and (c) absence of donor-specific antibody at time of transplant, within 30 days of the biopsy, and up to 4 measurements at later time points. ResultsThese stringent requirements identified 28 “pure” cases of late TCMR/BL. Low-grade glomerulitis, peritubular capillaritis, or chronic transplant glomerulopathy were found in 9/28 (32%) biopsies. Serum creatinine showed complete short-term remission in 7/10 (70%) BL and 9/18 (50%) TCMR patients 1 month postbiopsy. Yet, both treated and untreated patients demonstrated further decline in graft function as assessed by serum creatinine and estimated glomerular filtration rate. ConclusionsLate TCMR seen in 7.9% of biopsies can contribute to significant deterioration of graft function in patients in whom the dominant contribution of antibody-mediated injury has been reasonably excluded. Our data also reinforce existing literature showing that microvascular lesions do not have absolute specificity for a diagnosis of antibody-mediated rejection.

Evaluation of the gastrointestinal tract as potential route of primary polyomavirus infection in mice

Background Detection of Polyomavirus (PyV) DNA in metropolitan rivers worldwide has led to the suggestion that primary viral infection can occur by the oral route. The aim of this study was to test this notion experimentally. Methods Mouse PyV (MPyV) was used to infect C57BL/6J mice by the nasal or intragastric route. Viral load kinetics was studied 3, 7, 10, 14, 21 and 28 days post-infection (dpi) using quantitative PCR. Results Following nasal infection, MPyV DNA was readily detected in many organs including lung, heart, aorta, colon, and stool with viral loads in the range of 103–106 copies/mg wet weight that peaked 7–10 dpi. Complete viral clearance occurred in the serum and kidney by 28 dpi, while clearance in other organs was partial with a 10–100 fold decrease in viral load. In contrast, following intragastric infection peak detection of PyV was delayed to 21 dpi, and viral loads were up to 3 logs lower. There was no detectable virus in the heart, colon, or stool. Conclusions The intragastric route of MPyV infection is successful, not as efficacious as the respiratory route, and associated with delayed viral dissemination as well as a lower peak MPyV load in individual organs.