Achieving efficacy in subjects with sustained pegvaliase-neutralizing antibody responses.

Pegvaliase (Palynziq®) is an enzyme substitution therapy using PEGylated recombinant Anabaena variabilis phenylalanine ammonia lyase (PAL) to reduce blood phenylalanine (Phe) levels in adults with phenylketonuria (PKU). In Phase 3 clinical studies, all subjects treated with pegvaliase developed anti-drug antibodies. To specifically evaluate pegvaliase-neutralizing antibodies (NAbs) and assess impact on pegvaliase efficacy, a novel hybrid ligand-binding/tandem mass spectrometry NAb assay was developed. Analysis of Phase 3 study samples revealed that pegvaliase NAb titers developed during early treatment (≤6 months after treatment initiation), and then plateaued and persisted in the majority of subjects during late treatment (>6 months). Subjects with the lowest/undetectable NAb titers had relatively high plasma pegvaliase concentrations and experienced the most rapid decline in blood Phe concentrations at relatively low pegvaliase dose concentrations. In contrast, subjects with higher NAb titers generally had lower plasma pegvaliase concentrations on similar low doses, with little change in blood Phe concentrations. However, with additional time on treatment and individualized dose titration, the majority of subjects achieved substantial and sustained blood Phe reduction, including those with higher NAb titers. Moreover, after maturation of the anti-pegvaliase immune response, NAb titers were stable over time and did not rise in response to dose increases; thus, subjects did not require additional dose increases to maintain reduction in blood Phe.

A polyomavirus peptide binds to the capsid VP1 pore and has potent antiviral activity against BK and JC polyomaviruses.

In pursuit of therapeutics for human polyomaviruses, we identified a peptide derived from the BK polyomavirus (BKV) minor structural proteins VP2/3 that is a potent inhibitor of BKV infection with no observable cellular toxicity. The thirteen-residue peptide binds to major structural protein VP1 with single-digit nanomolar affinity. Alanine-scanning of the peptide identified three key residues, substitution of each of which results in ~1000 fold loss of binding affinity with a concomitant reduction in antiviral activity. Structural studies demonstrate specific binding of the peptide to the pore of pentameric VP1. Cell-based assays demonstrate nanomolar inhibition (EC50) of BKV infection and suggest that the peptide acts early in the viral entry pathway. Homologous peptide exhibits similar binding to JC polyomavirus VP1 and inhibits infection with similar potency to BKV in a model cell line. Lastly, these studies validate targeting the VP1 pore as a novel strategy for the development of anti-polyomavirus agents.

Human Memory B Cells Harbor Diverse Cross-Neutralizing Antibodies against BK and JC Polyomaviruses.

Human polyomaviruses cause a common childhood infection worldwide and typically elicit a neutralizing antibody and cellular immune response, while establishing a dormant infection in the kidney with minimal clinical manifestations. However, viral reactivation can cause severe pathology in immunocompromised individuals. We developed a high-throughput, functional antibody screen to examine the humoral response to BK polyomavirus. This approach enabled the isolation of antibodies from all peripheral B cell subsets and revealed the anti-BK virus antibody repertoire as clonally complex with respect to immunoglobulin sequences and isotypes (both IgM and IgG), including a high frequency of monoclonal antibodies that broadly neutralize BK virus subtypes and the related JC polyomavirus. Cryo-electron microscopy of a broadly neutralizing IgG single-chain variable fragment complexed with BK virus-like particles revealed the quaternary nature of a conserved viral epitope at the junction between capsid pentamers. These features unravel a potent modality for inhibiting polyomavirus infection in kidney transplant recipients and other immunocompromised patients.

JC polyoma viruria associates with protection from chronic kidney disease independently from apolipoprotein L1 genotype in African Americans.

Background: Viral infections can trigger chronic kidney disease (CKD) and the urine virome may inform risk. The Natural History of APOL1-Associated Nephropathy Study (NHAANS) reported that urine JC polyomavirus (JCPyV) associated with a lower risk of APOL1-associated nephropathy in African Americans. Herein, association was assessed between urine JCPyV with CKD in African Americans independent from the APOL1 genotype. Methods: Quantitative polymerase chain reaction was performed for urinary detection of JCPyV and BK polyoma virus (BKPyV) in 200 newly recruited nondiabetic African Americans. A combined analysis was performed in these individuals plus 300 NHAANS participants. Results: In the 200 new participants, urine JCPyV was present in 8.8% of CKD cases and 45.8% of nonnephropathy controls (P = 3.0 × 10-8). In those with APOL1 renal-risk genotypes, JCPyV was detected in 5.1% of cases and 40.0% of controls (P = 0.0002). In those lacking APOL1 renal-risk genotypes, JCPyV was detected in 12.2% of cases and 48.8% of controls (P = 8.5 × 10-5). BKPyV was detected in 1.3% of cases and 0.8% of controls (P = 0.77). In a combined analysis with 300 NHAANS participants (n = 500), individuals with urine JCPyV had a 63% lower risk of CKD compared with those without urine JCPyV (odds ratio 0.37; P = 4.6 × 10-6). RNA fluorescence in situ hybridization confirmed the presence of JCPyV genomic DNA and JCPyV messenger RNA (mRNA) in nondiseased kidney. Conclusions: Inverse relationships exist between JCPyV viruria and non-diabetic CKD. Future studies should determine whether renal inflammation associated with CKD is less permissive for JCPyV reactivation/replication or whether JCPyV is a marker of reduced host immune responsiveness that diminishes immune pathologic contributions to CKD.

Correlation of BK Virus Neutralizing Serostatus With the Incidence of BK Viremia in Kidney Transplant Recipients.

BACKGROUND: BK virus (BKV)-associated nephropathy is the second leading cause of graft loss in kidney transplant recipients. Due to the high prevalence of persistent infection with BKV in the general population, it is possible that either the transplant recipient or donor may act as the source of virus resulting in viruria and viremia. Although several studies suggest a correlation between donor-recipient serostatus and the development of BK viremia, specific risk factors for BKV-related complications in the transplant setting remain to be established. METHODS: We retrospectively determined the pretransplant BKV neutralizing serostatus of 116 donors (D)-recipient (R) pairs using infectious BKV neutralization assays with representatives from the 4 major viral serotypes. The neutralizing serostatus of donors and recipients was then correlated with the incidence of BK viremia during the first year posttransplantation. RESULTS: There were no significant differences in baseline demographics or transplant data among the 4 neutralizing serostatus groups, with the exception of calculated panel-reactive antibody which was lowest in the D+/R- group. Recipients of kidneys from donors with significant serum neutralizing activity (D+) had elevated risk for BK viremia, regardless of recipient serostatus (D+ versus D-: odd ratio, 5.0; 95% confidence interval, 1.9-12.7]; P = 0.0008). Furthermore, donor-recipient pairs with D+/R- neutralizing serostatus had the greatest risk for BK viremia (odds ratio, 4.9; 95% confidence interval, 1.7-14.6; P = 0.004). CONCLUSIONS: Donor neutralizing serostatus correlates significantly with incidence of posttransplant BK viremia. Determination of donor-recipient neutralizing serostatus may be useful in assessing the risk of BKV infection in kidney transplant recipients.

Kaposi's sarcoma-associated herpesvirus microRNAs target IRAK1 and MYD88, two components of the toll-like receptor/interleukin-1R signaling cascade, to reduce inflammatory-cytokine expression.

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is the causative agent of KS, an important AIDS-associated malignancy. KSHV expresses at least 18 different mature microRNAs (miRNAs). We identified interleukin-1 receptor (IL-1R)-associated kinase 1 (IRAK1) as a potential target of miR-K12-9 (miR-K9) in an array data set examining changes in cellular gene expression levels in the presence of KSHV miRNAs. Using 3'-untranslated region (3'UTR) luciferase reporter assays, we confirmed that miR-K9 and other miRNAs inhibit IRAK1 expression. In addition, IRAK1 expression is downregulated in cells transfected with miR-K9 and during de novo KSHV infection. IRAK1 is an important component of the Toll-like receptor (TLR)/IL-1R signaling cascade. The downregulation of IRAK1 by miR-K9 resulted in the decreased stimulation of NF-κB activity in endothelial cells treated with IL-1α and in B cells treated with a TLR7/8 agonist. Interestingly, miR-K9 had a greater effect on NF-κB activity than did a small interfering RNA (siRNA) targeting IRAK1 despite the more efficient downregulation of IRAK1 expression with the siRNA. We hypothesized that KSHV miRNAs may also be regulating a second component of the TLR/IL-1R signaling cascade, resulting in a stronger phenotype. Reanalysis of the array data set identified myeloid differentiation primary response protein 88 (MYD88) as an additional potential target. 3'UTR luciferase reporter assays and Western blot analysis confirmed the targeting of MYD88 by miR-K5. The presence of miR-K9 and miR-K5 inhibited the production of IL-6 and IL-8 upon the IL-1α stimulation of endothelial cells. These results demonstrate KSHV-encoded miRNAs regulating the TLR/IL-1R signaling cascade at two distinct points and suggest the importance of these pathways during viral infection.

A system for the analysis of BKV non-coding control regions: application to clinical isolates from an HIV/AIDS patient.

The human polyomavirus BK virus (BKV) is an important opportunistic pathogen whose disease prevalence continues to increase with the growing immunocompromised population. To date, the major determinant of replication in cell culture has not been formally proven. BKV exists as archetype virus and rearranged variants, which are classified based on the DNA sequence of their non-coding control regions (NCCRs). The archetype BKV NCCR is divided into five blocks of sequence and rearranged variants contain deletions and duplications of these blocks. In this study, a genetic system was developed and used to identify the major determinant of replication ability in primary renal proximal tubule epithelial cells, the natural host cell of BKV. This system was also used to analyze NCCR variants isolated from an immunocompromised patient which contain assorted rearrangement patterns and functional differences. This study solidifies the NCCR as the major genetic determinant of BKV replication ability in vitro.

Regulation of tumor necrosis factor-like weak inducer of apoptosis receptor protein (TWEAKR) expression by Kaposi's sarcoma-associated herpesvirus microRNA prevents TWEAK-induced apoptosis and inflammatory cytokine expression.

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is the causative agent of KS, the second most common AIDS-associated malignancy. KSHV expresses at least 18 different mature microRNAs (miRNAs) during latency. To identify cellular targets of KSHV miRNAs, we have analyzed a previously reported series of microarrays examining changes in cellular gene expression in the presence of KSHV miRNAs. Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) receptor (TWEAKR) was among the most consistently and robustly downregulated genes in the presence of KSHV miR-K12-10a (miR-K10a). Results from luciferase assays with reporter plasmids containing the 3' untranslated region (UTR) of TWEAKR suggest a targeting of TWEAKR by miR-K10a. The mutation of two predicted miR-K10a recognition sites within the 3' UTR of TWEAKR completely disrupts inhibition by miR-K10a. The expression of TWEAKR was downregulated in cells transfected with miR-K10a as well as during de novo KSHV infection. In a KS tumor-derived endothelial cell line, the downregulation of TWEAKR by miR-K10a resulted in reduced levels of TWEAK-induced caspase activation. In addition, cells transfected with miR-K10a showed less induction of apoptosis by annexin V staining and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assays. Finally, the downregulation of TWEAKR by miR-K10a in primary human endothelial cells resulted in a decrease in levels of expression of the proinflammatory cytokines interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) in response to TWEAK. These results identify and validate an important cellular target of KSHV miRNAs. Furthermore, we demonstrate that a viral miRNA protects cells from apoptosis and suppresses a proinflammatory response, which may have significant implications in the complex context of KS lesions.

Global effects of BKV infection on gene expression in human primary kidney epithelial cells.

BK virus (BKV) is a ubiquitous human pathogen that establishes a lifelong persistent infection in kidney epithelial cells. BKV reactivation within these cells results in a lytic infection in immunocompromised patients. Little is known about the specific interactions of BKV and the host cell during persistence and reactivation. We performed global cellular gene expression analyses using microarrays to characterize the global effect of BKV on primary kidney epithelial cells during the viral life cycle. Our results demonstrate that BKV primarily activates genes involved in cell cycle regulation and apoptosis (58% and 44% of upregulated genes at 48 and 72 h post-infection, respectively). Surprisingly, we observed that only four genes were downregulated during infection and that only two genes directly involved in the inflammatory response were differentially expressed. These results provide information about how BKV interacts with a cell type in which it both establishes persistence and undergoes lytic reactivation.

BK virus and human cancer: innocent until proven guilty.

BK virus (BKV) is a polyomavirus that ubiquitously infects the human population. Following a typically subclinical primary infection, BKV establishes a life-long persistent infection in the kidney and urinary tract. BKV is known to reactivate and cause severe disease in immunosuppressed patients, particularly renal and bone marrow transplant patients. Infection of BKV in rodent animal models or cells in culture often results in tumor formation or transformation, respectively. When co-expressed with activated oncogenes, BKV large tumor antigen drives the transformation of primary human cells. An etiological role of BKV in human cancer, however, remains controversial. Multiple reports have demonstrated conflicting results in regards to the presence of BKV sequences and/or proteins in various tumor types. This review compiles the most recent findings of BKV detection in a number of human cancers. Due to the lack of conclusive causality data from these studies, there does not appear to be a definitive association between BKV and human cancers.

A truncated T antigen expressed from an alternatively spliced BK virus early mRNA.

The early region of BK virus (BKV) is known to encode two well-characterized tumour (T) antigens, large T antigen (TAg) and small T antigen (tAg). In this study, we provide evidence of a third early BKV mRNA that codes for an additional early region product with an apparent molecular mass of 17-20 kDa. This truncated form of TAg (truncTAg) is expressed from an alternatively spliced mRNA that is derived from the excision of a second intron from the mRNA encoding TAg. The first 133 aa of truncTAg are identical to those of TAg but the additional splice results in translation from a different reading frame, adding three new amino acids before reaching a stop codon. TruncTAg is expressed in both BKV-transformed and lytically infected cells and it is found to be primarily localized to the nucleus. The function of BKV truncTAg is likely to be relevant to transformation, similar to the additional T antigens of simian virus 40, JC virus and mouse polyomavirus.

Restriction of human polyomavirus BK virus DNA replication in murine cells and extracts.

BK virus (BKV) causes persistent and asymptomatic infections in most humans and is the etiologic agent of polyomavirus-associated nephropathy (PVAN) and other pathologies. Unfortunately, there are no animal models with which to study activation of BKV replication in the human kidney and the accompanying PVAN. Here we report studies of the restriction of BKV replication in murine cells and extracts and the cause(s) of this restriction. Upon infection of murine cells, BKV expressed large T antigen (TAg), but viral DNA replication and progeny were not detected. Transfection of murine cells with BKV TAg expression vectors also caused TAg expression without accompanying DNA replication. Analysis of the replication of DNAs containing chimeric BKV and murine polyomavirus origins revealed the importance of BKV core origin sequences and TAg for DNA replication. A sensitive assay was developed with purified BKV TAg that supported TAg-dependent BKV DNA replication with human but not with murine cell extracts. Addition of human replication proteins, DNA polymerase alpha-primase, replication protein A, or topoisomerase I to the murine extracts with BKV TAg did not rescue viral DNA replication. Notably, addition of murine extracts to human extracts inhibited BKV TAg-dependent DNA replication at a step prior to or during unwinding of the viral origin. These findings and differences in replication specificity between BKV TAg and the TAgs of simian virus 40 (SV40) and JC virus (JCV) and their respective origins implicate features of the BKV TAg and origin distinct from SV40 and JCV in restriction of BKV replication in murine cells.

Early events during BK virus entry and disassembly.

BK virus (BKV) is a nonenveloped, ubiquitous human polyomavirus that establishes a persistent infection in healthy individuals. It can be reactivated, however, in immunosuppressed patients and cause severe diseases, including polyomavirus nephropathy. The entry and disassembly mechanisms of BKV are not well defined. In this report, we characterized several early events during BKV infection in primary human renal proximal tubule epithelial (RPTE) cells, which are natural host cells for BKV. Our results demonstrate that BKV infection in RPTE cells involves an acidic environment relatively early during entry, followed by transport along the microtubule network to reach the endoplasmic reticulum (ER). A distinct disulfide bond isomerization and cleavage pattern of the major capsid protein VP1 was observed, which was also influenced by alterations in pH and disruption of trafficking to the ER. A dominant negative form of Derlin-1, an ER protein required for retro-translocation of certain misfolded proteins, inhibited BKV infection. Consistent with this, we detected an interaction between Derlin-1 and VP1. Finally, we show that proteasome function is also linked to BKV infection and capsid rearrangement. These results indicate that BKV early entry and disassembly are highly regulated processes involving multiple cellular components.

The role of polyomaviruses in human disease.

The human polyomaviruses, BK virus and JC virus, have long been associated with serious diseases including polyomavirus nephropathy and progressive multifocal leukoencephalopathy. Both viruses establish ubiquitous, persistent infections in healthy individuals. Reactivation can occur when the immune system is impaired, leading to disease progression. Recently, the human polyomavirus family has expanded with the identification of three new viruses (KI, WU and Merkel cell polyomavirus), all of which may prove to be involved in human disease. This review describes the general aspects of human polyomavirus infections and pathogenicity. Current topics of investigation and future directions in the field are also discussed.

Transforming growth factor-beta-mediated regulation of BK virus gene expression.

The increasing prevalence of BK virus (BKV)-associated diseases in immunosuppressed patients has prompted an investigation of the immune response to BKV, especially the role of cytokines in regulating viral replication. We examined the effect of TGF-beta, a cytokine that is stimulated by certain immunosuppressive therapies, on BKV gene expression during lytic infection of renal proximal tubule epithelial cells. Viral gene expression, and specifically the activity of the BKV early promoter, is regulated by TGF-beta in a strain-dependent manner. Promoter activity is upregulated in the presence of TGF-beta for the TU strain of BKV, and not for the Dik, Dunlop, or Proto-2 strains. Using site-directed mutagenesis, we have identified a small segment of the TU promoter that is required for stimulation in response to TGF-beta. These results demonstrate that BKV strains can respond differently to cytokine treatment and suggest that TGF-beta may play a role in the reactivation of BKV.

Inhibitory effect of gamma interferon on BK virus gene expression and replication.

BK virus (BKV) is widely accepted to be the causative agent of polyomavirus nephropathy. In immunocompromised individuals, especially kidney transplant recipients, BKV can replicate in kidney epithelial cells, causing loss of renal function and eventual destruction of the graft. Advances in immunosuppressive therapies may be partially responsible for the increasing incidence of polyomavirus nephropathy among transplant recipients by more effectively eliminating components of the immune system, such as gamma interferon (IFN-gamma)-producing lymphocytes, that keep BKV infections at a subclinical level. In this study, we investigated the role of IFN-gamma in regulating lytic infection by BKV. Treatment with IFN-gamma inhibited the expression of the viral early protein large tumor antigen (TAg) and the late protein VP1 in a dose-dependent manner. We detected 1.6- and 12-fold reductions in TAg transcripts at 48 and 96 h postinfection, respectively, with 250 U/ml IFN-gamma, suggesting that IFN-gamma-mediated inhibition occurs at the level of transcription. Furthermore, IFN-gamma inhibited the level of viral progeny production as much as 50-fold at a multiplicity of infection (MOI) of 0.5 and 80-fold at an MOI of 0.1. The inhibitory effects of IFN-gamma were similar for three different strains of BKV examined. These results indicate an important role for IFN-gamma in regulating BKV lytic infection.

Analysis of the interaction of the adenovirus L1 52/55-kilodalton and IVa2 proteins with the packaging sequence in vivo and in vitro.

We previously showed that the adenovirus IVa2 and L1 52/55-kDa proteins interact in infected cells and the IVa2 protein is part of two virus-specific complexes (x and y) formed in vitro with repeated elements of the packaging sequence called the A1-A2 repeats. Here we demonstrate that both the IVa2 and L1 52/55-kDa proteins bind in vivo to the packaging sequence and that each protein-DNA interaction is independent of the other. There is a strong and direct interaction of the IVa2 protein with DNA in vitro. This interaction is observed when probes containing the A1-A2 or A4-A5 repeats are used, but it is not found by using an A5-A6 probe. Furthermore, we show that complex x is likely a heterodimer of IVa2 and an unknown viral protein, while complex y is a monomer or multimer of IVa2. No in vitro interaction of purified L1 52/55-kDa protein with the packaging sequence was found, suggesting that the L1 52/55-kDa protein-DNA interaction may be mediated by an intermediate protein. Results support roles for both the L1 52/55-kDa and IVa2 proteins in DNA encapsidation.

A peptide containing a novel FPGN CD40-binding sequence enhances adenoviral infection of murine and human dendritic cells.

CD40 is a receptor with numerous functions in the activation of antigen presenting cells (APCs), particularly dendritic cells (DC). Using phage display technology, we identified linear peptides containing a novel FPGN/S consensus sequence that enhances the binding of phage to a purified murine CD40-immunoglobulin (Ig) fusion protein (CD40-Ig), but not to Ig alone. To examine the ability the FPGN/S peptides to enhance adenoviral infection of CD40-positive cells, we used bifunctional peptides consisting of an FPGN-containing peptide covalently linked to an adenoviral knob-binding peptide (KBP). One of these, FPGN2-KBP, was able to enhance adenoviral infection of both murine and human DCs in a dose-dependent manner. FPGN2-KBP also improved infection of murine B cell blasts, a murine B lymphoma cell line (L10A), and immortalized human B cells. To demonstrate that enhancement of adenoviral infection depended on the presence of CD40, we analyzed infection of the breast cancer line, SKBR3, that does not express CD40 or the adenovirus cellular receptor, CAR. Infection of SKBR3 cells was enhanced by FPGN2-KBP following transient transfection with a plasmid vector that expresses murine CD40, but not when the cells were mock-transfected. In conclusion, we have isolated a peptide that binds to murine CD40, and promotes the uptake of adenoviruses into CD40-expressing cells of both murine and human origin, suggesting that it may have potential applications for antigen delivery to CD40-positive antigen-presenting cells.

Engineered fibronectin type III domain with a RGDWXE sequence binds with enhanced affinity and specificity to human alphavbeta3 integrin.

Fibronectin is an extracellular matrix protein with broad binding specificity to cell surface receptors, integrins. The tenth fibronectin type III domain (FNfn10) is a small, autonomous domain of fibronectin containing the RGE sequence that is directly involved in integrin binding. However, in isolation FNfn10 only weakly bind to integrins. We reasoned that high-affinity and high-specificity variants of FNfn10 to a particular integrin could be engineered by optimizing residues surrounding the integrin-binding RGD sequence in the flexible FG loop. Affinity maturation of FNfn10 to alphavbeta3 integrin, an integrin up-regulated in angiogenic endothelial cells and in some metastatic tumor cells, yielded alphavbeta3-binding FNfn10 mutants with a novel RGDWXE consensus sequence. We characterized one of the RGDWXE-modified clones, FNfn10-3JCLI4, as purified protein. FNfn10-3JCLI4 binds with high affinity and specificity to purified alphavbeta3 integrin. Alanine scanning mutagenesis suggested that both the tryptophan and glutamic acid residues following the RGD sequence are required for maximal affinity and specificity for alphavbeta3. FNfn10-3JCLI4 specifically stained alphavbeta3-positive cells as detected with flow cytometry and it inhibited alphavbeta3-dependent cell adhesion. As with the anti-alphavbeta3 antibody LM609, FNfn10-3JCLI4 can interfere with in vitro capillary formation. Taken together, these data show that FNfn10-3JCL14 is a specific, high-affinity alphavbeta3-binding protein that can inhibit alphavbeta3-dependent cellular processes similar to an anti-alphavbeta3 monoclonal antibody. These properties, combined with the small, monomeric, cysteine-free and highly stable structure of FNfn10-3JCLI4, may make this protein useful in future applications involving detection and targeting of alphavbeta3-positive cells.