Margot Brickelmaier

Structural and Functional Differences Between Glycosylated and Non-glycosylated Forms of Human Interferon-β (IFN-β)

AbstractPurpose. Two recombinant IFN-β products have been approved for the treatment of multiple sclerosis, a glycosylated form with the predicted natural amino acid sequence (IFN-β-la) and a non-glycosylated form that has a Met-1 deletion and a Cys-17 to Ser mutation (IFN-β-lb). The structural basis for activity differences between IFN-β-la and IFN-β-lb, is determined. Methods. In vitro antiviral, antiproliferative and immunomodulatory assays were used to directly compare the two IFN-β products. Size exclusion chromatography (SEC), SDS-PAGE, thermal denaturation, and X-ray crystallography were used to examine structural differences. Results. IFN-β- la was 10 times more active than IFN-β- Ib with specific activities in a standard antiviral assay of 20 × 107 lU/mg for IFN-β-la and 2 × 107 lU/mg for IFN-β-lb. Of the known structural differences between IFN-β-la and IFN-β-lb, only glycosylation affected in vitro activity. Deglycosylation of IFN-β-la produced a decrease in total activity that was primarily caused by the formation of an insoluble disulfide-linked IFN precipitate. Deglycosylation also resulted in an increased sensitivity to thermal denaturation. SEC data for IFN-β-lb revealed large, soluble aggregates that had reduced antiviral activity (approximated at 0.7 × 107 lU/mg). Crystallographic data for IFN-β-la revealed that the glycan formed H-bonds with the peptide backbone and shielded an uncharged surface from solvent exposure. Conclusions. Together these results suggest that the greater biological activity of IFN-β-la is due to a stabilizing effect of the carbohydrate on structure.

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.

Alefacept, an Immunomodulatory Recombinant LFA-3/IgG1 Fusion Protein, Induces CD16 Signaling and CD2/CD16-Dependent Apoptosis of CD2+ Cells

Alefacept, an immunomodulatory recombinant fusion protein composed of the first extracellular domain of LFA-3 fused to the human IgG1 hinge, CH2, and CH3 domains, has recently been shown in phase II and III clinical trials to safely reduce disease expression in patients with chronic plaque psoriasis. Alefacept modulates the function of and selectively induces apoptosis of CD2+ human memory-effector T cells in vivo. We have sought to gain further understanding of the mechanisms of action that influence the biological activity of alefacept and may contribute to its efficacy and patient responsiveness. Specifically evaluated is the ability of alefacept to activate intracellular signals mediated via CD2 and/or FcγRIII (CD16). Experimentation using isoforms of alefacept engineered to have amino acid substitutions in the IgG1 CH2 domain that impact FcγR binding indicate that alefacept mediates cognate interactions between cells expressing human CD2 and CD16 to activate cells, e.g., increase extracellular signal-regulated kinase phosphorylation, up-regulate cell surface expression of the activation marker CD25, and induce release of granzyme B. In the systems used, this signaling is shown to require binding to CD2 and CD16 and be mediated through CD16, but not CD2. Experimentation using human CD2-transgenic mice and isoforms of alefacept confirmed the requirement for FcγR binding for detection of the pharmacological effects of alefacept in vivo. Thus alefacept acts as an effector molecule, mediating cognate interactions to activate FcγR+ cells (e.g., NK cells) to induce apoptosis of sensitive CD2+ target cells.

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.

Inflammatory arthritis can be reined in by CpG-induced DC-NK cell cross talk

Unmethylated CpG-oligodeoxynucleotides (ODNs) are generally thought of as potent adjuvants with considerable therapeutic potential to enhance immune responses against microbes and tumors. Surprisingly, certain so-called stimulatory CpG-ODNs strongly inhibited the effector phase of inflammatory arthritis in the K/BxN serum transfer system, either preventively or therapeutically. Also unexpected was that the inhibitory influence did not depend on the adaptive immune system cells mobilized in an immunostimulatory context. Instead, they relied on cells of the innate immune system, specifically on cross talk between CD8α+ dendritic cells and natural killer cells, resulting in suppression of neutrophil recruitment to the joint, orchestrated through interleukin-12 and interferon-γ. These findings highlight potential applications of CpG-ODNs and downstream molecules as antiinflammatory agents.

Comparison of Gene Expression Patterns Induced by Treatment of Human Umbilical Vein Endothelial Cells with IFN-α2b vs. IFN-β1a: Understanding the Functional Relationship Between Distinct Type I Interferons That Act Through a Common Receptor

We analyzed whether interferon-alpha 2b (IFN-alpha 2b) and IFN-beta 1a engage their common receptor to generate activated receptor complexes possessing distinct signaling properties. Human vascular endothelial cells (HUVEC) are 100-1000-fold more sensitive to IFN-beta 1a than to IFN-alpha 2b in in vitro assays. An nonarray-based expression profiling (GeneCalling) technology was employed to compare the patterns and levels of gene expression induced by these IFN as the broadest means by which signaling events could be measured. To distinguish subtype-related differences from dose-related effects, RNA was prepared from HUVEC treated with 50-5000 pg/ml of each IFN. The results showed that at 50 pg/ml IFN, only a subset of the genes induced by IFN-beta 1a were also induced by IFN-alpha 2b and that individual genes were induced to higher levels by IFN-beta 1a. In contrast, at 5000 pg/ml, both subtypes induced essentially identical sets of genes to similar levels of expression. No genes were seen to be induced uniquely by IFN-alpha 2b but not by IFN-beta 1a. The results show that the two IFN are intrinsically capable of inducing similar gene induction responses and do not provide evidence that they generate activated receptor complexes possessing distinct signaling properties. In contrast, the two IFN generate gene induction patterns that are both qualitatively and quantitatively distinct at subsaturating and potentially physiologically more relevant concentrations.

PEGylated Interferon Beta-1a: Meeting an Unmet Medical Need in the Treatment of Relapsing Multiple Sclerosis

Multiple sclerosis is a chronic autoimmune disease of the central nervous system for which a number of disease-modifying therapies are available, including interferon beta (Avonex®, Rebif®, and Betaseron/Betaferon®), glatiramer acetate (Copaxone®), and an anti-VLA4 monoclonal antibody (Tysabri®). Despite the availability and efficacy of these protein and peptide drugs, there remains a significant number of patients who are untreated, including those with relatively mild disease who choose not to initiate therapy, those wary of injections or potential adverse events associated with therapy, and those who have stopped therapy due to perceived lack of efficacy. Since these drugs have side effects that may affect a patients decision to initiate and to remain on treatment, there is a need to provide a therapy that is safe and efficacious but that requires a reduced dosing frequency and hence a concomitant reduction in the frequency of side effects. Here we describe the development of a PEGylated form of interferon beta-1a that is currently being tested in a multicenter, randomized, double-blind, parallel-group, placebo-controlled study in relapsing multiple sclerosis patients, with the aim of determining the safety and efficacy of 125 microg administered via the subcutaneous route every 2 or 4 weeks.

Recombinant ST2 boosts hepatic Th2 response in vivo

Excessive scarring or fibrosis is a common feature of a wide spectrum of diseases characterized by an exaggerated Th2 response. The TLR/IL‐1 receptor (IL‐1R)‐related protein ST2 is expressed in a membrane‐bound form selectively by Th2 cells and was shown to be indispensable for some in vivo Th2 responses. ST2 was also found to block TLR signaling. We addressed the impact of the ST2 pathway on fibrogenesis using a mouse model of hepatic injury and fibrosis induced by carbon tetrachloride (CCl4). We showed that cytokine production by intrahepatic lymphocytes from CCl4‐injured liver is abrogated in the absence of TLR‐4. Interfering with the ST2 pathway using an ST2‐Fc fusion protein accelerated and enhanced hepatic fibrosis, paralleled by the increasing ex vivo secretion of Th2 cytokines IL‐4, ‐5, ‐10, and ‐13 by intrahepatic lymphocytes of ST2‐Fc‐treated, CCl4‐gavaged mice. Absence of IL‐4/13 signaling in IL‐4Rα‐deficient mice obliterated this ST2‐Fc effect on fibrogenesis. Moreover, depletion of CD4+ T cells abrogated ST2‐Fc‐enhanced Th2 cytokines and accelerated fibrosis. Thus, ST2‐Fc caused overproduction of Th2 cytokines by intrahepatic CD4+ T cells, possibly by modifying TLR‐4 signaling in injured liver. This ST2‐Fc‐driven Th2 response exacerbated CCl4‐induced hepatic fibrosis.

Cytotoxicity of Combinations of IFN-β and Chemotherapeutic Drugs

Interferon-β (IFN-β) induces various antiproliferative activities. In solid tumor cells, IFN-β inhibits cell cycle progression, which mainly occurs as S phase accumulation. The IFN-β-induced cell cycle effect has been implicated in the antitumor effect of combinations of IFN-β and chemotherapeutic drugs. In this report, we characterized the viability of various human tumor cells in vitro after combination treatment with IFN-β protein and the chemotherapeutic drugs, cis-platinum (II) diamine dichloride (cisplatin), 5-fluorouracil (5-FU), paclitaxel (Taxol) and gemcitabine. IFN-β could significantly potentiate the cytotoxicity of these chemotherapeutic drugs. The potentiating effect was observed after pretreatment of tumor cells with IFN-β but did not require the constant presence of IFN-β. The potentiating effect correlated with the sensitivity of the tumor cells to the IFN-β-induced cytotoxicity. Furthermore, chemotherapeutic drugs also potentiated the cytotoxicity of IFN-β. We conclude that the cell cycl...

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.