Kenneth J. Simon

Anti-JC Virus Antibodies: Implications for PML Risk Stratification

A study was undertaken to establish an enzyme‐linked immunosorbent assay (ELISA) to detect JC virus (JCV)‐specific antibodies in multiple sclerosis (MS) patients, and to evaluate its potential utility for identifying patients at higher or lower risk (ie, risk stratification) of developing progressive multifocal leukoencephalopathy (PML).

Identification and Characterization of Mefloquine Efficacy against JC Virus In Vitro

ABSTRACT Progressive multifocal leukoencephalopathy (PML) is a rare but frequently fatal disease caused by the uncontrolled replication of JC virus (JCV), a polyomavirus, in the brains of some immunocompromised individuals. Currently, no effective antiviral treatment for this disease has been identified. As a first step in the identification of such therapy, we screened the Spectrum collection of 2,000 approved drugs and biologically active molecules for their anti-JCV activities in an in vitro infection assay. We identified a number of different drugs and compounds that had significant anti-JCV activities at micromolar concentrations and lacked cellular toxicity. Of the compounds with anti-JCV activities, only mefloquine, an antimalarial agent, has been reported to show sufficiently high penetration into the central nervous system such that it would be predicted to achieve efficacious concentrations in the brain. Additional in vitro experiments demonstrated that mefloquine inhibits the viral infection rates of three different JCV isolates, JCV(Mad1), JCV(Mad4), and JCV(M1/SVEΔ), and does so in three different cell types, transformed human glial (SVG-A) cells, primary human fetal glial cells, and primary human astrocytes. Using quantitative PCR to quantify the number of viral copies in cultured cells, we have also shown that mefloquine inhibits viral DNA replication. Finally, we demonstrated that mefloquine does not block viral cell entry; rather, it inhibits viral replication in cells after viral entry. Although no suitable animal model of PML or JCV infection is available for the testing of mefloquine in vivo, our in vitro results, combined with biodistribution data published in the literature, suggest that mefloquine could be an effective therapy for PML.

Affinity enhancement of an in vivo matured therapeutic antibody using structure-based computational design

Improving the affinity of a high‐affinity protein–protein interaction is a challenging problem that has practical applications in the development of therapeutic biomolecules. We used a combination of structure‐based computational methods to optimize the binding affinity of an antibody fragment to the I‐domain of the integrin VLA1. Despite the already high affinity of the antibody (Kd ∼7 nM) and the moderate resolution (2.8 Å) of the starting crystal structure, the affinity was increased by an order of magnitude primarily through a decrease in the dissociation rate. We determined the crystal structure of a high‐affinity quadruple mutant complex at 2.2 Å. The structure shows that the design makes the predicted contacts. Structural evidence and mutagenesis experiments that probe a hydrogen bond network illustrate the importance of satisfying hydrogen bonding requirements while seeking higher‐affinity mutations. The large and diverse set of interface mutations allowed refinement of the mutant binding affinity prediction protocol and improvement of the single‐mutant success rate. Our results indicate that structure‐based computational design can be successfully applied to further improve the binding of high‐affinity antibodies.

Progressive Multifocal Leukoencephalopathy (PML) Development Is Associated With Mutations in JC Virus Capsid Protein VP1 That Change Its Receptor Specificity

Progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease caused by JC virus (JCV) infection of oligodendrocytes, may develop in patients with immune disorders following reactivation of chronic benign infection. Mutations of JCV capsid viral protein 1 (VP1), the capsid protein involved in binding to sialic acid cell receptors, might favor PML onset. Cerebrospinal fluid sequences from 37/40 PML patients contained one of several JCV VP1 amino acid mutations, which were also present in paired plasma but not urine sequences despite the same viral genetic background. VP1-derived virus-like particles (VLPs) carrying these mutations lost hemagglutination ability, showed different ganglioside specificity, and abolished binding to different peripheral cell types compared with wild-type VLPs. However, mutants still bound brain-derived cells, and binding was not affected by sialic acid removal by neuraminidase. JCV VP1 substitutions are acquired intrapatient and might favor JCV brain invasion through abrogation of sialic acid binding with peripheral cells, while maintaining sialic acid-independent binding with brain cells.

Adaptive Mutations in the JC Virus Protein Capsid Are Associated with Progressive Multifocal Leukoencephalopathy (PML)

PML is a progressive and mostly fatal demyelinating disease caused by JC virus infection and destruction of infected oligodendrocytes in multiple brain foci of susceptible individuals. While JC virus is highly prevalent in the human population, PML is a rare disease that exclusively afflicts only a small percentage of immunocompromised individuals including those affected by HIV (AIDS) or immunosuppressive drugs. Viral- and/or host-specific factors, and not simply immune status, must be at play to account for the very large discrepancy between viral prevalence and low disease incidence. Here, we show that several amino acids on the surface of the JC virus capsid protein VP1 display accelerated evolution in viral sequences isolated from PML patients but not in sequences isolated from healthy subjects. We provide strong evidence that at least some of these mutations are involved in binding of sialic acid, a known receptor for the JC virus. Using statistical methods of molecular evolution, we performed a comprehensive analysis of JC virus VP1 sequences isolated from 55 PML patients and 253 sequences isolated from the urine of healthy individuals and found that a subset of amino acids found exclusively among PML VP1 sequences is acquired via adaptive evolution. By modeling of the 3-D structure of the JC virus capsid, we showed that these residues are located within the sialic acid binding site, a JC virus receptor for cell infection. Finally, we go on to demonstrate the involvement of some of these sites in receptor binding by demonstrating a profound reduction in hemagglutination properties of viral-like particles made of the VP1 protein carrying these mutations. Collectively, these results suggest that a more virulent PML causing phenotype of JC virus is acquired via adaptive evolution that changes viral specificity for its cellular receptor(s).

Antibody-mediated blockade of integrin ?v?6 inhibits tumor progression in vivo by a transforming growth factor-?–regulated mechanism

The alpha(v)beta(6) integrin is up-regulated on epithelial malignancies and has been implicated in various aspects of cancer progression. Immunohistochemical analysis of alpha(v)beta(6) expression in 10 human tumor types showed increased expression relative to normal tissues. Squamous carcinomas of the cervix, skin, esophagus, and head and neck exhibited the highest frequency of expression, with positive immunostaining in 92% (n = 46), 84% (n = 49), 68% (n = 56), and 64% (n = 100) of cases, respectively. We studied the role of alpha(v)beta(6) in Detroit 562 human pharyngeal carcinoma cells in vitro and in vivo. Prominent alpha(v)beta(6) expression was detected on tumor xenografts at the tumor-stroma interface resembling the expression on human head and neck carcinomas. Nonetheless, coculturing cells in vitro with matrix proteins did not up-regulate alpha(v)beta(6) expression. Detroit 562 cells showed alpha(v)beta(6)-dependent adhesion and activation of transforming growth factor-beta (TGF-beta) that was inhibited >90% with an alpha(v)beta(6) blocking antibody, 6.3G9. Although both recombinant soluble TGF-beta receptor type-II (rsTGF-beta RII-Fc) and 6.3G9 inhibited TGF-beta-mediated Smad2/3 phosphorylation in vitro, there was no effect on proliferation. Conversely, in vivo, 6.3G9 and rsTGF-beta RII-Fc inhibited xenograft tumor growth by 50% (n = 10, P < 0.05) and >90% (n = 10, P < 0.001), respectively, suggesting a role for the microenvironment in this response. However, stromal collagen and smooth muscle actin content in xenograft sections were unchanged with treatments. Although further studies are required to consolidate in vitro and in vivo results and define the mechanisms of tumor inhibition by alpha(v)beta(6) antibodies, our findings support a role for alpha(v)beta(6) in human cancer and underscore the therapeutic potential of function blocking alpha(v)beta(6) antibodies.

Sequencing and Analysis of JC Virus DNA From Natalizumab-Treated PML Patients

Background. Progressive multifocal leukoencephalopathy (PML) in natalizumab-treated MS patients is linked to JC virus (JCV) infection. JCV sequence variation and rearrangements influence viral pathogenicity and tropism. To better understand PML development, we analyzed viral DNA sequences in blood, CSF and/or urine of natalizumab-treated PML patients. Methods. Using biofluid samples from 17 natalizumab-treated PML patients, we sequenced multiple isolates of the JCV noncoding control region (NCCR), VP1 capsid coding region, and the entire 5 kb viral genome. Results. Analysis of JCV from multiple biofluids revealed that individuals were infected with a single genotype. Across our patient cohort, multiple PML-associated NCCR rearrangements and VP1 mutations were present in CSF and blood, but absent from urine-derived virus. NCCR rearrangements occurred in CSF of 100% of our cohort. VP1 mutations were observed in blood or CSF in 81% of patients. Sequencing of complete JCV genomes demonstrated that NCCR rearrangements could occur without VP1 mutations, but VP1 mutations were not observed without NCCR rearrangement. Conclusions. These data confirm that JCV in natalizumab-PML patients is similar to that observed in other PML patient groups, multiple genotypes are associated with PML, individual patients appear to be infected with a single genotype, and PML-associated mutations arise in patients during PML development.

Multi-site analytical validation of an assay to detect anti-JCV antibodies in human serum and plasma

BACKGROUND JC virus (JCV) infection is a prerequisite for development of progressive multifocal leukoencephalopathy (PML). We previously described the development of a novel, two-step enzyme-linked immunosorbent assay (ELISA) that detects anti-JCV antibodies in human serum or plasma, and the potential clinical utility of anti-JCV antibody status for PML risk stratification. OBJECTIVES To validate the anti-JCV antibody ELISA at multiple clinical laboratories in order to demonstrate the robustness of the method. STUDY DESIGN Analytical validation of the ELISA was performed at four laboratories by evaluation of intra- and inter-assay precision, analytical specificity and sensitivity, matrix interference, robustness, sample and reagent stability. RESULTS Analytical validation demonstrated that the assay is sensitive, specific, and precise. The assay sensitivity was estimated at 1.7ng/mL using a humanized anti-JCV monoclonal antibody control. The sensitivity to detect JCV infection was estimated to be 97.5%. The specificity of the assay to discriminate JCV-specific antibodies from antibodies directed to BK virus, a related polyomavirus, was also demonstrated. The inter- and intra-assay precision was ≤6.0% and 9.8% for the screening step and 2.6% and 11.3% for the confirmation step. Results obtained for plasma and serum were highly congruent, and assay robustness was demonstrated by the highly concordant results generated by four laboratories testing a common panel of 100 blinded samples. CONCLUSIONS The novel, two-step ELISA to detect anti-JCV antibodies in human serum and plasma is robust, and assay performance is consistent and reproducible in multiple laboratories, making it suitable to support testing for global clinical studies.

Distribution and Quantity of Sites of John Cunningham Virus Persistence in Immunologically Healthy Patients: Correlation With John Cunningham Virus Antibody and Urine John Cunningham Virus DNA

Importance Although seroepidemiological studies indicate that greater than 50% of the population has been infected with John Cunningham virus (JCV), the sites of JCV persistence remain incompletely characterized. Objective To determine sites of JCV persistence in immunologically healthy individuals. Design, Setting, and Participants Tissue specimens from multiple sites including brain, renal, and nonrenal tissues were obtained at autopsy performed in the Department of Pathology at the University of Kentucky from 12 immunologically healthy patients between February 9, 2011, and November 27, 2012. Quantitative polymerase chain reaction was performed on the tissue specimens and urine. Serum JCV antibody status was determined by enzyme-linked immunosorbent assay. Main Outcomes and Measures The detection and quantification of JCV from the tissues by quantitative polymerase chain reaction illuminated sites of viral persistence. These results were correlated with JCV antibody levels. Results Autopsies were performed on 12 individuals, 10 men and 2 women, ranging in age from 25 to 75 years (mean, 55.3 years). Seven of 12 individuals were JCV antibody seropositive based on absorbance units. Serostatus was associated with amounts of JCV DNA in urine and its tissue distribution. John Cunningham virus DNA was found in 75% of genitourinary tissue samples from donors (18 of 24) with high JCV antibody levels, 13.3% of donors with low levels i(4 of 30), and 0% of seronegative persons (0 of 32). In nongenitourinary tissues, JCV DNA was detected in 45.1% of tissue samples of donors (32 of 71) with high JCV, 2.2% of donors with low JCV serostatus (2 of 93), and 0% of seronegative persons (0 of 43). Genitourinary tissues had higher copy numbers than other sites. John Cunningham virus DNA was detected in urine of seronegative individuals in a research-grade assay. Conclusions and Relevance Persistent (latent or actively replicating) JCV infection mostly predominates in genitourinary tissues but distributes in other tissues at low copy number. The distribution and copy numbers of the virus appear to correlate with urinary JCV shedding and serostatus.

Synthetic antibodies and peptides recognizing progressive multifocal leukoencephalopathy-specific point mutations in polyomavirus JC capsid viral protein 1

Polyomavirus JC (JCV) is the causative agent of progressive multifocal leukoencephalopathy (PML), a rare and frequently fatal brain disease that afflicts a small fraction of the immune-compromised population, including those affected by AIDS and transplantation recipients on immunosuppressive drug therapy. Currently there is no specific therapy for PML. The major capsid viral protein 1 (VP1) involved in binding to sialic acid cell receptors is believed to be a key player in pathogenesis. PML-specific mutations in JCV VP1 sequences present at the binding pocket of sialic acid cell receptors, such as L55F and S269F, abolish sialic acid recognition and might favor PML onset. Early diagnosis of these PML-specific mutations may help identify patients at high risk of PML, thus reducing the risks associated with immunosuppressive therapy. As a first step in the development of such early diagnostic tools, we report identification and characterization of affinity reagents that specifically recognize PML-specific mutations in VP1 variants using phage display technology. We first identified 2 peptides targeting wild type VP1 with moderate specificity. Fine-tuning via selection of biased libraries designed based on 2 parental peptides yielded peptides with different, yet still moderate, bindinspecificities. In contrast, we had great success in identifying synthetic antibodies that recognize one of the PML-specific mutations (L55F) with high specificity from the phage-displayed libraries. These peptides and synthetic antibodies represent potential candidates for developing tailored immune-based assays for PML risk stratification in addition to complementing affinity reagents currently available for the study of PML and JCV.