Pharmacokinetics/pharmacodynamics of benralizumab in chronic rhinosinusitis with nasal polyps: Phase III, randomized, placebo-controlled OSTRO trial.

AIMS: Benralizumab, a humanized, afucosylated monoclonal antibody against the interleukin 5 receptor, α subunit, causes rapid depletion of eosinophils by antibody-dependent cellular cytotoxicity. We investigated the pharmacokinetic and pharmacodynamic effects of benralizumab in patients with chronic rhinosinusitis with nasal polyps (CRSwNP) from the phase III OSTRO trial. METHODS: Patients received a placebo or 30 mg of benralizumab by subcutaneous injection every 8 weeks (first three doses every 4 weeks) to week 48; a subset of patients continued in an extended follow-up period to assess treatment durability to week 80. Serum benralizumab concentrations and blood eosinophil and basophil counts were assessed to week 80. Biomarker assessments were performed on nasal polyp tissue biopsies at week 56 and nasal lining fluid at weeks 24 and 56 to examine changes in immune cells and inflammatory mediators. RESULTS: Among 185 patients in this analysis, 93 received benralizumab. Serum benralizumab concentrations reached a steady state by week 24 (median concentration 385.52 ng mL-1); blood eosinophils were almost fully depleted and blood basophils were reduced between weeks 16 and 56. Nasal polyp tissue eosinophils decreased with benralizumab from 57.6 cells mm-2 at baseline to 0 cells mm-2 at week 56 (P < .001 vs placebo), and tissue mast cells were numerically reduced. In nasal lining fluid, eosinophil-derived neurotoxin was significantly reduced at weeks 24 and 56 (P < .001) and interleukin-17 at week 56 (P < .05) with benralizumab. CONCLUSION: Benralizumab treatment led to rapid, sustained, nearly complete depletion of eosinophils from blood and nasal polyp tissue in patients with CRSwNP.

Humanised effector-null FcγRIIA antibody inhibits immune complex-mediated proinflammatory responses.

OBJECTIVE: Immune complexes (ICs) play a critical role in the pathology of autoimmune diseases. The aim of this study was to generate and characterise a first-in-class anti-FcγRIIA antibody (Ab) VIB9600 (previously known as MEDI9600) that blocks IgG immune complex-mediated cellular activation for clinical development. METHODS: VIB9600 was humanised and optimised from the IV.3 Ab. Binding affinity and specificity were determined by Biacore and ELISA. Confocal microscopy, Flow Cytometry-based assays and binding competition assays were used to assess the mode of action of the antibody. In vitro cell-based assays were used to demonstrate suppression of IC-mediated inflammatory responses. In vivo target suppression and efficacy was demonstrated in FcγRIIA-transgenic mice. Single-dose pharmacokinetic (PK)/pharmacodynamic study multiple dose Good Laboratory Practice (GLP) toxicity studies were conducted in non-human primates. RESULTS: We generated a humanised effector-deficient anti-FcγRIIA antibody (VIB9600) that potently blocks autoantibody and IC-mediated proinflammatory responses. VIB9600 suppresses FcγRIIA activation by blocking ligand engagement and by internalising FcγRIIA from the cell surface. VIB9600 inhibits IC-induced type I interferons from plasmacytoid dendritic cells (involved in SLE), antineutrophil cytoplasmic antibody (ANCA)-induced production of reactive oxygen species by neutrophils (involved in ANCA-associated vasculitis) and IC-induced tumour necrosis factor α and interleukin-6 production (involved in rheumatoid arthritis). In FcγRIIA transgenic mice, VIB9600 suppressed antiplatelet antibody-induced thrombocytopaenia, acute anti-GBM Ab-induced nephritis and anticollagen Ab-induced arthritis. VIB9600 also exhibited favourable PK and safety profiles in cynomolgus monkey studies. CONCLUSIONS: VIB9600 is a specific humanised antibody antagonist of FcγRIIA with null effector function that warrants further clinical development for the treatment of IC-mediated diseases.

Vaccination with synthetic long peptide formulated with CpG in an oil-in-water emulsion induces robust E7-specific CD8 T cell responses and TC-1 tumor eradication.

BACKGROUND: Despite considerable efforts at developing therapeutic vaccines for cancer, clinical translation of preclinical successes has been challenging, largely due to the difficulty of inducing strong and sustained cytotoxic T lymphocyte (CTL) responses in patients. Several peptide-based cancer vaccines have failed to show sustainable tumor regression in the clinic, possibly because of a lack of optimization of both the adjuvant and antigen components of the preparations. Here, we aimed to develop and optimize a vaccine format utilizing a synthetic long peptide (SLP) containing the human papilloma virus 16 (HPV16) E7 antigen, with a centrally located defined MHC class I epitope, and evaluate its immunogenicity and efficacy in combination with various adjuvant formulations. METHODS: E731-73 SLP was tested alone or in combination with toll-like receptor (TLR)3, TLR4, TLR7/8 and TLR9 agonists and formulated in oil-in-water (o/w) or water-in-oil (w/o) emulsions to determine a vaccine format inducing a robust CD8 T cell response in murine models. Once a lead vaccine format was determined, we examined its ability to inhibit tumor growth in the murine TC-1 model that expresses HPV16 E7 antigen. RESULTS: We identified the TLR9 agonist CpG formulated in a squalene-based o/w emulsion as the most potent adjuvant, inducing the expansion of multifunctional antigen specific CD8 T cells with cytolytic potential. We also demonstrated that SLP E731-73 + CpG + o/w emulsion vaccine can provide prophylactic and more importantly, therapeutic benefit in the TC-1 murine tumor model. CONCLUSIONS: Our results demonstrate that the novel vaccine format E7 SLP + CpG delivered in an o/w emulsion holds potential for the promotion of strong CTL responses and tumor eradication and encourages further development of peptide/adjuvant vaccines in cancer immunotherapy strategies.

[18F]-Fluorodeoxyglucose Uptake in Lymphoid Tissue Serves as a Predictor of Disease Outcome in the Nonhuman Primate Model of Monkeypox Virus Infection.

Real-time bioimaging of infectious disease processes may aid countermeasure development and lead to an improved understanding of pathogenesis. However, few studies have identified biomarkers for monitoring infections using in vivo imaging. Previously, we demonstrated that positron emission tomography/computed tomography (PET/CT) imaging with [18F]-fluorodeoxyglucose (FDG) can monitor monkeypox disease progression in vivo in nonhuman primates (NHPs). In this study, we investigated [18F]-FDG-PET/CT imaging of immune processes in lymphoid tissues to identify patterns of inflammation in the monkepox NHP model and to determine the value of [18F]-FDG-PET/CT as a biomarker for disease and treatment outcomes. Quantitative analysis of [18F]-FDG-PET/CT images revealed differences between moribund and surviving animals at two sites vital to the immune response to viral infections, bone marrow and lymph nodes (LNs). Moribund NHPs demonstrated increased [18F]-FDG uptake in bone marrow 4 days postinfection compared to surviving NHPs. In surviving, treated NHPs, increase in LN volume correlated with [18F]-FDG uptake and peaked 10 days postinfection, while minimal lymphadenopathy and higher glycolytic activity were observed in moribund NHPs early in infection. Imaging data were supported by standard virology, pathology, and immunology findings. Even with the limited number of subjects, imaging was able to differentiate the difference between disease outcomes, warranting additional studies to demonstrate whether [18F]-FDG-PET/CT can identify other, subtler effects. Visualizing altered metabolic activity at sites involved in the immune response by [18F]-FDG-PET/CT imaging is a powerful tool for identifying key disease-specific time points and locations that are most relevant for pathogenesis and treatment.IMPORTANCE Positron emission tomography and computed tomography (PET/CT) imaging is a universal tool in oncology and neuroscience. The application of this technology to infectious diseases is far less developed. We used PET/CT imaging with [18F]-labeled fluorodeoxyglucose ([18F]-FDG) in monkeys after monkeypox virus exposure to monitor the immune response in lymphoid tissues. In lymph nodes of surviving monkeys, changes in [18F]-FDG uptake positively correlated with enlargement of the lymph nodes and peaked on day 10 postinfection. In contrast, the bone marrow and lymph nodes of nonsurvivors showed increased [18F]-FDG uptake by day 4 postinfection with minimal lymph node enlargement, indicating that elevated cell metabolic activity early after infection is predictive of disease outcome. [18F]-FDG-PET/CT imaging can provide real-time snapshots of metabolic activity changes in response to viral infections and identify key time points and locations most relevant for monitoring the development of pathogenesis and for potential treatment to be effective.

Targeting the GM-CSF receptor for the treatment of CNS autoimmunity.

In multiple sclerosis (MS), there is a growing interest in inhibiting the pro-inflammatory effects of granulocyte-macrophage colony-stimulating factor (GM-CSF). We sought to evaluate the therapeutic potential and underlying mechanisms of GM-CSF receptor alpha (Rα) blockade in animal models of MS. We show that GM-CSF signaling inhibition at peak of chronic experimental autoimmune encephalomyelitis (EAE) results in amelioration of disease progression. Similarly, GM-CSF Rα blockade in relapsing-remitting (RR)-EAE model prevented disease relapses and inhibited T cell responses specific for both the inducing and spread myelin peptides, while reducing activation of mDCs and inflammatory monocytes. In situ immunostaining of lesions from human secondary progressive MS (SPMS), but not primary progressive MS patients shows extensive recruitment of GM-CSF Rα+ myeloid cells. Collectively, this study reveals a pivotal role of GM-CSF in disease relapses and the benefit of GM-CSF Rα blockade as a potential novel therapeutic approach for treatment of RRMS and SPMS.

Scientific and Regulatory Policy Committee (SRPC) paper: validation of digital pathology systems in the regulated nonclinical environment.

Digital Pathology Systems (DPS) are dynamic, image-based computer systems that enable the acquisition, management, and interpretation of pathology information generated from digitized glass slides. This article provides a roadmap for (1) qualification of a whole slide scanner (WSS) during a validation project, (2) validation of software required to generate the whole slide image (WSI), and (3) an introduction to visual digital image evaluation and image analysis. It describes a validation approach that can be utilized when validating a DPS. It is not the intent of this article to provide guidance on when validation of DPS is required. Rather, the article focuses on technical aspects of validation of the WSS system (WSS, IT infrastructure, and associated software) portion of a DPS and covers the processes of setting up the WSS for scanning a glass slide through saving a WSI on a server. Validation of a computerized system, such as a DPS, for use in a regulated nonclinical environment is governed by Code of Federal Regulations (CFR) Title 21 part 11: Electronic Records; Electronic Signature and predicate rules associated with Good Laboratory Practices documents including 21 CFR part 58. Similar regulation and predicate rules apply in the European Union and Japan.

Urinary markers differentially associate with kidney inflammatory activity and chronicity measures in patients with lupus nephritis.

OBJECTIVE: Lupus nephritis (LN) is diagnosed by biopsy, but longitudinal monitoring assessment methods are needed. Here, in this preliminary and hypothesis-generating study, we evaluate the potential for using urine proteomics as a non-invasive method to monitor disease activity and damage. Urinary biomarkers were identified and used to develop two novel algorithms that were used to predict LN activity and chronicity. METHODS: Baseline urine samples were collected for four cohorts (healthy donors (HDs, n=18), LN (n=42), SLE (n=17) or non-LN kidney disease biopsy control (n=9)), and over 1 year for patients with LN (n=42). Baseline kidney biopsies were available for the LN (n=46) and biopsy control groups (n=9). High-throughput proteomics platforms were used to identify urinary analytes ≥1.5 SD from HD means, which were subjected to stepwise, univariate and multivariate logistic regression modelling to develop predictive algorithms for National Institutes of Health Activity Index (NIH-AI)/National Institutes of Health Chronicity Index (NIH-CI) scores. Kidney biopsies were analysed for macrophage and neutrophil markers using immunohistochemistry (IHC). RESULTS: In total, 112 urine analytes were identified from LN, SLE and biopsy control patients as both quantifiable and overexpressed compared with HDs. Regression analysis identified proteins associated with the NIH-AI (n=30) and NIH-CI (n=26), with four analytes common to both groups, demonstrating a difference in the mechanisms associated with NIH-AI and NIH-CI. Pathway analysis of the NIH-AI and NIH-CI analytes identified granulocyte-associated and macrophage-associated pathways, and the presence of these cells was confirmed by IHC in kidney biopsies. Four markers each for the NIH-AI and NIH-CI were identified and used in the predictive algorithms. The NIH-AI algorithm sensitivity and specificity were both 93% with a false-positive rate (FPR) of 7%. The NIH-CI algorithm sensitivity was 88%, specificity 96% and FPR 4%. The accuracy for both models was 93%. CONCLUSIONS: Longitudinal predictions suggested that patients with baseline NIH-AI scores of ≥8 were most sensitive to improvement over 6-12 months. Viable approaches such as this may enable the use of urine samples to monitor LN over time.

Correction: Upregulation of annexin A1 protein expression in the intratumoral vasculature of human non-small-cell lung carcinoma and rodent tumor models.

[This corrects the article DOI: 10.1371/journal.pone.0234268.].

Upregulation of annexin A1 protein expression in the intratumoral vasculature of human non-small-cell lung carcinoma and rodent tumor models.

Annexin A1 (anxA1) is an immunomodulatory protein that has been proposed as a tumor vascular target for antitumor biologic agents, yet to date the vascular expression of anxA1 in specific tumor indications has not been systematically assessed. Attempts to evaluate vascular anxA1 expression by immunohistochemistry are complicated by a lack of available antibodies that are both specific for anxA1 and bind the N-terminal-truncated form of anxA1 that has previously been identified in tumor vasculature. To study the vascular expression pattern of anxA1 in non-small-cell lung carcinoma (NSCLC), we isolated an antibody capable of binding N-terminal-truncated anxA127-346 and employed it in immunohistochemical studies of human lung specimens. Lung tumor specimens evaluated with this antibody revealed vascular (endothelial) anxA1 expression in five of eight tumor samples studied, but no vascular anxA1 expression was observed in normal lung tissue. Tumor microarray analysis further demonstrated positive vascular staining for anxA1 in 30 of 80 NSCLC samples, and positive staining of neoplastic cells was observed in 54 of 80 samples. No correlation was observed between vascular and parenchymal anxA1 expression. Two rodent tumor models, B16-F10 and Py230, were determined to have upregulated anxA1 expression in the intratumoral vasculature. These data validate anxA1 as a potential vascular anti-tumor target in a subset of human lung tumors and identify rodent models which demonstrate anxA1 expression in tumor vasculature.

Safety and Clinical Activity of MEDI1873, a Novel GITR Agonist, in Advanced Solid Tumors.

PURPOSE: The safety and preliminary efficacy of MEDI1873, an agonistic IgG1 fusion protein targeting glucocorticoid-induced TNF receptor-related protein (GITR), were evaluated in an open-label, first-in-human, phase I, dose escalation study in previously treated patients with advanced solid tumors. PATIENTS AND METHODS: Two single-patient cohorts at 1.5 and 3 mg i.v. were followed by 3+3 dose escalation in six cohorts at 7.5, 25, 75, 250, 500, and 750 mg, all every 2 weeks, for up to 52 weeks. Primary endpoints were safety and tolerability, dose-limiting toxicities (DLT), and MTD. Secondary endpoints included antitumor activity, pharmacokinetics, immunogenicity, and pharmacodynamics. RESULTS: Forty patients received MEDI1873. Three experienced DLTs: grade 3 worsening tumor pain (250 mg); grade 3 nausea, vomiting, and headache (500 mg); and grade 3 non-ST segment elevation myocardial infarction (750 mg). An MTD was not reached and treatment was well tolerated up to 500 mg. Most common treatment-related adverse events were headache (25%), infusion-related reaction (17.5%), and decreased appetite (17.5%). MEDI1873 exposure was dose proportional. Antidrug-antibody incidence was low. MEDI1873 increased peripheral CD4+ effector memory T-cell proliferation as well as cytokines associated with effector T-cell activation at dose levels ≥75 mg. The best response was stable disease (SD) in 17 patients (42.5%), including 1 unconfirmed partial response. Eight patients (20.0%) had SD ≥24 weeks. CONCLUSIONS: MEDI1873 showed acceptable safety up to 500 mg i.v. every 2 weeks with pharmacodynamics activity, and prolonged SD in some patients. However, further development is not planned because of lack of demonstrated tumor response.

Pancreatic ß-Cell Rest Replenishes Insulin Secretory Capacity and Attenuates Diabetes in an Extreme Model of Obese Type 2 Diabetes.

The onset of common obesity-linked type 2 diabetes (T2D) is marked by exhaustive failure of pancreatic ß-cell functional mass to compensate for insulin resistance and increased metabolic demand, leading to uncontrolled hyperglycemia. Here, the ß-cell-deficient obese hyperglycemic/hyperinsulinemic KS db/db mouse model was used to assess consequential effects on ß-cell functional recovery by lowering glucose homeostasis and/or improving insulin sensitivity after treatment with thiazolidinedione therapy or glucagon-like peptide 1 receptor agonism alone or in combination with sodium/glucose cotransporter 2 inhibition (SGLT-2i). SGLT-2i combination therapies improved glucose homeostasis, independent of changes in body weight, resulting in a synergistic increase in pancreatic insulin content marked by significant recovery of the ß-cell mature insulin secretory population but with limited changes in ß-cell mass and no indication of ß-cell dedifferentiation. Restoration of ß-cell insulin secretory capacity also restored biphasic insulin secretion. These data emphasize that by therapeutically alleviating the demand for insulin in vivo, irrespective of weight loss, endogenous ß-cells recover significant function that can contribute to attenuating diabetes. Thus, this study provides evidence that alleviation of metabolic demand on the ß-cell, rather than targeting the ß-cell itself, could be effective in delaying the progression of T2D.

Both diet and Helicobacter pylori infection contribute to atherosclerosis in pre- and postmenopausal cynomolgus monkeys.

A number of viruses and bacterial species have been implicated as contributors to atherosclerosis, potentially providing novel pathways for prevention. Epidemiological studies examining the association between Helicobacter pylori and cardiovascular disease have yielded variable results and no studies have been conducted in nonhuman primates. In this investigation, we examined the relationship between H. pylori infection and atherosclerosis development in socially housed, pre- and postmenopausal cynomolgus macaques consuming human-like diets. Ninety-four premenopausal cynomolgus monkeys (Macaca fascicularis) were fed for 36 months an atherogenic diet deriving its protein from either casein lactalbumin(CL) or high isoflavone soy (SOY). Animals were then ovariectomized and fed either the same or the alternate diet for an additional 36 months. Iliac artery biopsies were obtained at the time of ovariectomy and iliac and coronary artery sections were examined at the end of the study. Evidence of H. pylori infection was found in 64% of the monkeys and 46% of animals had live H. pylori within coronary atheromas as determined by mRNA-specific in situ hybridization. There was a significant linear relationship between the densities of gastric and atheroma organisms. Helicobactor pylori infection correlated with increased intimal plaque area and thickness at both the premenopausal and postmenopausal time points and regardless of diet (p< 0.01), although animals consuming the SOY diet throughout had the least amount of atherosclerosis. Additionally, plasma lipid profiles, intimal collagen accumulation, ICAM-1, and plaque macrophage densities were adversely affected by H. pylori infection among animals consuming the CL diet, while the SOY diet had the opposite effect. Plaque measurements were more highly associated with the densities of cagA-positive H. pylori within coronary atheromas than with the densities of gastric organisms, whereas plasma lipid changes were associated with H. pylori infection, but not cagA status. This study provides strong evidence that live H. pylori infects atheromas, exacerbates atherosclerotic plaque development, and alters plasma lipid profiles independently of diet or hormonal status. Finally, socially subordinate animals relative to their dominant counterparts had a greater prevalence of H. pylori, suggesting a stress effect. The results indicate that early H. pylori eradication could prevent or delay development of cardiovascular disease.

Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens.

Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of tissue samples from formalin-fixed, paraffin-embedded (FFPE) biopsies has become a key tool for understanding the complexity of tumor immunology and discovering novel predictive biomarkers for cancer immunotherapy. Immunoprofiling analysis of tissues requires the evaluation of combined markers, including inflammatory cell subpopulations and immune checkpoints, in the tumor microenvironment. The advent of novel multiplex immunohistochemical methods allows for a more efficient multiparametric analysis of single tissue sections than does standard monoplex immunohistochemistry (IHC). One commercially available multiplex immunofluorescence (IF) method is based on tyramide-signal amplification and, combined with multispectral microscopic analysis, allows for a better signal separation of diverse markers in tissue. This methodology is compatible with the use of unconjugated primary antibodies that have been optimized for standard IHC on FFPE tissue samples. Herein we describe in detail an automated protocol that allows multiplex IF labeling of carcinoma tissue samples with a six-marker multiplex antibody panel comprising PD-L1, PD-1, CD68, CD8, Ki-67, and AE1/AE3 cytokeratins with 4',6-diamidino-2-phenylindole as a nuclear cell counterstain. The multiplex panel protocol is optimized in an automated IHC stainer for a staining time that is shorter than that of the manual protocol and can be directly applied and adapted by any laboratory investigator for immuno-oncology studies on human FFPE tissue samples. Also described are several controls and tools, including a drop-control method for fine quality control of a new multiplex IF panel, that are useful for the optimization and validation of the technique.

Targeted drug delivery via caveolae-associated protein PV1 improves lung fibrosis.

Systemic administration of bio-therapeutics can result in only a fraction of drug reaching targeted tissues, with the majority of drug being distributed to tissues irrelevant to the drug's site of action. Targeted delivery to specific organs may allow for greater accumulation, better efficacy, and improved safety. We investigated how targeting plasmalemma vesicle-associated protein (PV1), a protein found in the endothelial caveolae of lungs and kidneys, can promote accumulation in these organs. Using ex vivo fluorescence imaging, we show that intravenously administered αPV1 antibodies localize to mouse lungs and kidneys. In a bleomycin-induced idiopathic pulmonary fibrosis (IPF) mouse model, αPV1 conjugated to Prostaglandin E2 (PGE2), a known anti-fibrotic agent, significantly reduced collagen content and fibrosis whereas a non-targeted PGE2 antibody conjugate failed to slow fibrosis progression. Our results demonstrate that PV1 targeting can be utilized to deliver therapeutics to lungs and this approach is potentially applicable for various lung diseases.

Neurturin and a GLP-1 Analogue Act Synergistically to Alleviate Diabetes in Zucker Diabetic Fatty Rats.

Neurturin (NRTN), a member of the glial-derived neurotrophic factor family, was identified from an embryonic chicken pancreatic cDNA library in a screen for secreted factors. In this study, we assessed the potential antidiabetic activities of NRTN relative to liraglutide, a glucagon-like peptide 1 receptor agonist, in Zucker diabetic fatty (ZDF) rats. Subcutaneous administration of NRTN to 8-week-old male ZDF rats prevented the development of hyperglycemia and improved metabolic parameters similar to liraglutide. NRTN treatment increased pancreatic insulin content and ß-cell mass and prevented deterioration of islet organization. However, unlike liraglutide-treated rats, NRTN-mediated improvements were not associated with reduced body weight or food intake. Acute NRTN treatment did not activate c-Fos expression in key feeding behavior and metabolic centers in ZDF rat brain or directly enhance glucose-stimulated insulin secretion from pancreatic ß-cells. Treating 10-week-old ZDF rats with sustained hyperglycemia with liraglutide resulted in some alleviation of hyperglycemia, whereas NRTN was not as effective despite improving plasma lipids and fasting glucose levels. Interestingly, coadministration of NRTN and liraglutide normalized hyperglycemia and other metabolic parameters, demonstrating that combining therapies with distinct mechanism(s) can alleviate advanced diabetes. This emphasizes that therapeutic combinations can be more effective to manage diabetes in individuals with uncontrolled hyperglycemia.

MEDI1873, a potent, stabilized hexameric agonist of human GITR with regulatory T-cell targeting potential.

Glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) is part of a system of signals involved in controlling T-cell activation. Targeting and agonizing GITR in mice promotes antitumor immunity by enhancing the function of effector T cells and inhibiting regulatory T cells. Here, we describe MEDI1873, a novel hexameric human GITR agonist comprising an IgG1 Fc domain, a coronin 1A trimerization domain and the human GITRL extracellular domain (ECD). MEDI1873 was optimized through systematic testing of different trimerization domains, aglycosylation of the GITRL ECD and comparison of different Fc isotypes. MEDI1873 exhibits oligomeric heterogeneity and superiority to an anti-GITR antibody with respect to evoking robust GITR agonism, T-cell activation and clustering of Fc gamma receptors. Further, it recapitulates, in vitro, several aspects of GITR targeting described in mice, including modulation of regulatory T-cell suppression and the ability to increase the CD8+:CD4+ T-cell ratio via antibody-dependent T-cell cytotoxicity. To support translation into a therapeutic setting, we demonstrate that MEDI1873 is a potent T-cell agonist in vivo in non-human primates, inducing marked enhancement of humoral and T-cell proliferative responses against protein antigen, and demonstrate the presence of GITR- and FoxP3-expressing infiltrating lymphocytes in a range of human tumors. Overall our data provide compelling evidence that MEDI1873 is a novel, potent GITR agonist with the ability to modulate T-cell responses, and suggest that previously described GITR biology in mice may translate to the human setting, reinforcing the potential of targeting the GITR pathway as a therapeutic approach to cancer.

Effects of soy isoflavones and conjugated equine estrogens on inflammatory markers in atherosclerotic, ovariectomized monkeys.

The effects of dietary soy isoflavones (IF) and conjugated equine estrogens (CEE) on circulating inflammatory markers were determined at the end of a 3-yr study of ovariectomized monkeys consuming a moderately atherogenic diet. Treatments were: 1) control, receiving alcohol-extracted soy-protein-based diet with low IF content (comparable to approximately 5 mg/d); 2) CEE, added to the control diet at a dose comparable to 0.625 mg/d; and 3) IF, consumed as a part of unextracted soy protein isolate at a dose comparable to 129 mg/d. Serum soluble vascular cell adhesion molecule-1 (sVCAM-1) was reduced by both IF (P < 0.006) and CEE (P < 0.0001) relative to controls. Serum monocyte chemoattractant protein (MCP)-1 was reduced by CEE (P < 0.0001) but not by IF (P = 1.00). Treatments did not affect serum IL-6 (P = 0.40), soluble E-selectin (P = 0.17), or C-reactive protein (P = 0.15). Serum MCP-1 and, to a lesser extent, IL-6 significantly correlated with atherosclerosis (plaque area) in the iliac and carotid arteries (all P < 0.05). Serum MCP-1 was also strongly associated with coronary artery atherosclerosis and with indices of plaque inflammation and matrix remodeling (matrix metalloproteinase-9) in the coronary artery intima (all P < 0.01). We conclude that, in this well-established nonhuman primate model of atherosclerosis, this dose of soy IF provided an antiinflammatory effect specific for sVCAM-1, whereas the effects of CEE extended to both sVCAM-1 and MCP1. It is possible that the atheroprotective effects of IF and CEE are mediated, at least in part, by effects on VCAM-1. The sites of IF inhibitory effects on sVCAM-1 production are not known, but likely candidates include the liver and/or the cardiovascular system.

A replication-deficient rabies virus vaccine expressing Ebola virus glycoprotein is highly attenuated for neurovirulence.

We are developing inactivated and live-attenuated rabies virus (RABV) vaccines expressing Ebola virus (EBOV) glycoprotein for use in humans and endangered wildlife, respectively. Here, we further characterize the pathogenesis of the live-attenuated RABV/EBOV vaccine candidates in mice in an effort to define their growth properties and potential for safety. RABV vaccines expressing GP (RV-GP) or a replication-deficient derivative with a deletion of the RABV G gene (RVΔG-GP) are both avirulent after intracerebral inoculation of adult mice. Furthermore, RVΔG-GP is completely avirulent upon intracerebral inoculation of suckling mice unlike parental RABV vaccine or RV-GP. Analysis of RVΔG-GP in the brain by quantitative PCR, determination of virus titer, and immunohistochemistry indicated greatly restricted virus replication. In summary, our findings indicate that RV-GP retains the attenuation phenotype of the live-attenuated RABV vaccine, and RVΔG-GP would appear to be an even safer alternative for use in wildlife or consideration for human use.

Cowpox virus infection of cynomolgus macaques as a model of hemorrhagic smallpox.

Hemorrhagic smallpox was a rare but severe manifestation of variola virus infection that resulted in nearly 100% mortality. Here we describe intravenous (IV) inoculation of cowpox virus Brighton Red strain in cynomolgus macaques (Macaca fascicularis) which resulted in disease similar in presentation to hemorrhagic smallpox in humans. IV inoculation of macaques resulted in a uniformly lethal disease within 12 days post-inoculation in two independent experiments. Clinical observations and hematological and histopathological findings support hemorrhagic disease. Cowpox virus replicated to high levels in blood (8.0-9.0 log(10) gene copies/mL) and tissues including lymph nodes, thymus, spleen, bone marrow, and lungs. This unique model of hemorrhagic orthopoxvirus infection provides an accessible means to further study orthopoxvirus pathogenesis and to identify virus-specific and nonspecific therapies. Such studies will serve to complement the existing nonhuman primate models of more classical poxviral disease.

Progression of pathogenic events in cynomolgus macaques infected with variola virus.

Smallpox, caused by variola virus (VARV), is a devastating human disease that affected millions worldwide until the virus was eradicated in the 1970 s. Subsequent cessation of vaccination has resulted in an immunologically naive human population that would be at risk should VARV be used as an agent of bioterrorism. The development of antivirals and improved vaccines to counter this threat would be facilitated by the development of animal models using authentic VARV. Towards this end, cynomolgus macaques were identified as adequate hosts for VARV, developing ordinary or hemorrhagic smallpox in a dose-dependent fashion. To further refine this model, we performed a serial sampling study on macaques exposed to doses of VARV strain Harper calibrated to induce ordinary or hemorrhagic disease. Several key differences were noted between these models. In the ordinary smallpox model, lymphoid and myeloid hyperplasias were consistently found whereas lymphocytolysis and hematopoietic necrosis developed in hemorrhagic smallpox. Viral antigen accumulation, as assessed immunohistochemically, was mild and transient in the ordinary smallpox model. In contrast, in the hemorrhagic model antigen distribution was widespread and included tissues and cells not involved in the ordinary model. Hemorrhagic smallpox developed only in the presence of secondary bacterial infections - an observation also commonly noted in historical reports of human smallpox. Together, our results support the macaque model as an excellent surrogate for human smallpox in terms of disease onset, acute disease course, and gross and histopathological lesions.