PET imaging of TSPO expression in immune cells can assess organ-level pathophysiology in high-consequence viral infections.

Ebola virus (EBOV) disease is characterized by lymphopenia, breach in vascular integrity, cytokine storm, and multiorgan failure. The pathophysiology of organ involvement, however, is incompletely understood. Using [18F]-DPA-714 positron emission tomography (PET) imaging targeting the translocator protein (TSPO), an immune cell marker, we sought to characterize the progression of EBOV-associated organ-level pathophysiology in the EBOV Rhesus macaque model. Dynamic [18F]-DPA-714 PET/computed tomography imaging was performed longitudinally at baseline and at multiple time points after EBOV inoculation, and distribution volumes (Vt) were calculated as a measure of peripheral TSPO binding. Using a mixed-effect linear regression model, spleen and lung Vt decreased, while the bone marrow Vt increased over time after infection. No clear trend was found for liver Vt. Multiple plasma cytokines correlated negatively with lung/spleen Vt and positively with bone marrow Vt. Multiplex immunofluorescence staining in spleen and lung sections confirmed organ-level lymphoid and monocytic loss/apoptosis, thus validating the imaging results. Our findings are consistent with EBOV-induced progressive monocytic and lymphocytic depletion in the spleen, rather than immune activation, as well as depletion of alveolar macrophages in the lungs, with inefficient reactive neutrophilic activation. Increased bone marrow Vt, on the other hand, suggests hematopoietic activation in response to systemic immune cell depletion and leukocytosis and could have prognostic relevance. In vivo PET imaging provided better understanding of organ-level pathophysiology during EBOV infection. A similar approach can be used to delineate the pathophysiology of other systemic infections and to evaluate the effectiveness of newly developed treatment and vaccine strategies.

Filoviruses Infect Rhesus Macaque Synoviocytes in Vivo and Primary Human Synoviocytes in Vitro.

The most commonly reported symptom of post-Ebola virus disease syndrome in survivors is arthralgia, yet involvement of the joints in acute or convalescent Ebola virus infection is not well characterized in human patients or animal models. Through immunohistochemistry, we found that the lining synovial intima of the stifle (knee) is a target for acute infection by Ebola virus/Kikwit, Ebola virus/Makona-C05, and Marburg virus/Angola in the rhesus macaque model. Furthermore, histologic analysis, immunohistochemistry, RNAscope in situ hybridization, and transmission electron microscopy showed that synoviocytes of the stifle, shoulder, and hip are a target for mouse-adapted Ebola virus/Yambuku-Mayinga infection during acute disease in rhesus macaques. A time course of infection study with Ebola virus/Kikwit found that the large joint synovium became immunopositive beginning on postinfection day 6. In total, the synovium of 28 of 30 rhesus macaques with terminal filovirus disease had evidence of infection (64 of 96 joints examined). On the basis of immunofluorescence, infected cell types included CD68+ type A (macrophage-like) synoviocytes and CD44+ type B (fibroblast-like) synoviocytes. Cultured primary human fibroblast-like synoviocytes were permissive to infection with Ebola and Marburg viruses in vitro. Because synovial joints include immune privileged sites, these findings are significant for future investigations of filovirus pathogenesis and persistence as well as arthralgias in acute and convalescent filovirus disease.

A Recombinant Rabies Virus Expressing the Marburg Virus Glycoprotein Is Dependent upon Antibody-Mediated Cellular Cytotoxicity for Protection against Marburg Virus Disease in a Murine Model.

Marburg virus (MARV) is a filovirus related to Ebola virus (EBOV) associated with human hemorrhagic disease. Outbreaks are sporadic and severe, with a reported case mortality rate of upward of 88%. There is currently no antiviral or vaccine available. Given the sporadic nature of outbreaks, vaccines provide the best approach for long-term control of MARV in regions of endemicity. We have developed an inactivated rabies virus-vectored MARV vaccine (FILORAB3) to protect against Marburg virus disease. Immunogenicity studies in our labs have shown that a Th1-biased seroconversion to both rabies virus and MARV glycoproteins (GPs) is beneficial for protection in a preclinical murine model. As such, we adjuvanted FILORAB3 with glucopyranosyl lipid adjuvant (GLA), a Toll-like receptor 4 agonist, in a squalene-in-water emulsion. Across two different BALB/c mouse challenge models, we achieved 92% protection against murine-adapted Marburg virus (ma-MARV). Although our vaccine elicited strong MARV GP antibodies, it did not strongly induce neutralizing antibodies. Through both in vitro and in vivo approaches, we elucidated a critical role for NK cell-dependent antibody-mediated cellular cytotoxicity (ADCC) in vaccine-induced protection. Overall, these findings demonstrate that FILORAB3 is a promising vaccine candidate for Marburg virus disease.IMPORTANCE Marburg virus (MARV) is a virus similar to Ebola virus and also causes a hemorrhagic disease which is highly lethal. In contrast to EBOV, only a few vaccines have been developed against MARV, and researchers do not understand what kind of immune responses are required to protect from MARV. Here we show that antibodies directed against MARV after application of our vaccine protect in an animal system but fail to neutralize the virus in a widely used virus neutralization assay against MARV. This newly discovered activity needs to be considered more when analyzing MARV vaccines or infections.

Persistence of Lassa Virus Associated With Severe Systemic Arteritis in Convalescing Guinea Pigs (Cavia porcellus).

Lassa fever (LF) survivors develop various clinical manifestations including polyserositis, myalgia, epididymitis, and hearing loss weeks to months after recovery from acute infection. We demonstrate a systemic lymphoplasmacytic and histiocytic arteritis and periarteritis in guinea pigs more than 2 months after recovery from acute Lassa virus (LASV) infection. LASV was detected in the arterial tunica media smooth muscle cells by immunohistochemistry, in situ hybridization, and transmission electron microscopy. Our results suggest that the sequelae of LASV infection may be due to virus persistence resulting in systemic vascular damage. These findings shed light on the pathogenesis of LASV sequelae in convalescent human survivors.

Ebola Virus Localization in the Macaque Reproductive Tract during Acute Ebola Virus Disease.

Sexual transmission of Ebola virus (EBOV) has been demonstrated more than a year after recovery from the acute phase of Ebola virus disease (EVD). The mechanisms underlying EBOV persistence and sexual transmission are not currently understood. Using the acute macaque model of EVD, we hypothesized EBOV would infect the reproductive tissues and sought to localize the infection in these tissues using immunohistochemistry and transmission electron microscopy. In four female and eight male macaques that succumbed to EVD between 6 and 9 days after EBOV challenge, we demonstrate widespread EBOV infection of the interstitial tissues and endothelium in the ovary, uterus, testis, seminal vesicle, epididymis, and prostate gland, with minimal associated tissue immune response or organ pathology. Given the widespread involvement of EBOV in the reproductive tracts of both male and female macaques, it is reasonable to surmise that our understanding of the mechanisms underlying sexual transmission of EVD and persistence of EBOV in immune-privileged sites would be facilitated by the development of a nonhuman primate model in which the macaques survived past the acute stage into convalescence.

An Inactivated Rabies Virus-Based Ebola Vaccine, FILORAB1, Adjuvanted With Glucopyranosyl Lipid A in Stable Emulsion Confers Complete Protection in Nonhuman Primate Challenge Models.

The 2013-2016 West African Ebola virus (EBOV) disease outbreak was the largest filovirus outbreak to date. Over 28 000 suspected, probable, or confirmed cases have been reported, with a 53% case-fatality rate. The magnitude and international impact of this EBOV outbreak has highlighted the urgent need for a safe and efficient EBOV vaccine. To this end, we demonstrate the immunogenicity and protective efficacy of FILORAB1, a recombinant, bivalent, inactivated rabies virus-based EBOV vaccine, in rhesus and cynomolgus monkeys. Our results demonstrate that the use of the synthetic Toll-like receptor 4 agonist glucopyranosyl lipid A in stable emulsion (GLA-SE) as an adjuvant increased the efficacy of FILORAB1 to 100% protection against lethal EBOV challenge, with no to mild clinical signs of disease. Furthermore, all vaccinated subjects developed protective anti-rabies virus antibody titers. Taken together, these results support further development of FILORAB1/GLA-SE as an effective preexposure EBOV vaccine.

Exposure of rhesus monkeys to cowpox virus Brighton Red by large-particle aerosol droplets results in an upper respiratory tract disease.

We previously demonstrated that small-particle (0.5-3.0 µm) aerosol infection of rhesus monkeys (Macaca mulatta) with cowpox virus (CPXV)-Brighton Red (BR) results in fulminant respiratory tract disease characterized by severe lung parenchymal pathology but only limited systemic virus dissemination and limited classic epidermal pox-like lesion development (Johnson et al., 2015). Based on these results, and to further develop CPXV as an improved model of human smallpox, we evaluated a novel large-particle aerosol (7.0-9.0 µm) exposure of rhesus monkeys to CPXV-BR and monitored for respiratory tract disease by serial computed tomography (CT). As expected, the upper respiratory tract and large airways were the major sites of virus-induced pathology following large-particle aerosol exposure. Large-particle aerosol CPXV exposure of rhesus macaques resulted in severe upper airway and large airway pathology with limited systemic dissemination.

Excess variants in AFF2 detected by massively parallel sequencing of males with autism spectrum disorder.

Autism spectrum disorder (ASD) is a heterogeneous disorder with substantial heritability, most of which is unexplained. ASD has a population prevalence of one percent and affects four times as many males as females. Patients with fragile X E (FRAXE) intellectual disability, which is caused by a silencing of the X-linked gene AFF2, display a number of ASD-like phenotypes. Duplications and deletions at the AFF2 locus have also been reported in cases with moderate intellectual disability and ASD. We hypothesized that other rare X-linked sequence variants at the AFF2 locus might contribute to ASD. We sequenced the AFF2 genomic region in 202 male ASD probands and found that 2.5% of males sequenced had missense mutations at highly conserved evolutionary sites. When compared with the frequency of missense mutations in 5545 X chromosomes from unaffected controls, we saw a statistically significant enrichment in patients with ASD (OR: 4.9; P < 0.014). In addition, we identified rare AFF2 3' UTR variants at conserved sites which alter gene expression in a luciferase assay. These data suggest that rare variation in AFF2 may be a previously unrecognized ASD susceptibility locus and may help explain some of the male excess of ASD.

Magnetic Resonance Imaging for Monitoring of Hepatic Disease Induced by Ebola Virus: a Nonhuman Primate Proof-of-Concept Study.

Severe liver impairment is a well-known hallmark of Ebola virus disease (EVD). However, the role of hepatic involvement in EVD progression is understudied. Medical imaging in established animal models of EVD (e.g., nonhuman primates [NHPs]) can be a strong complement to traditional assays to better investigate this pathophysiological process in vivo and noninvasively. In this proof-of-concept study, we used longitudinal multiparametric magnetic resonance imaging (MRI) to characterize liver morphology and function in nine rhesus monkeys after exposure to Ebola virus (EBOV). Starting 5 days postexposure, MRI assessments of liver appearance, morphology, and size were consistently compatible with the presence of hepatic edema, inflammation, and congestion, leading to significant hepatomegaly at necropsy. MRI performed after injection of a hepatobiliary contrast agent demonstrated decreased liver signal on the day of euthanasia, suggesting progressive hepatocellular dysfunction and hepatic secretory impairment associated with EBOV infection. Importantly, MRI-assessed deterioration of biliary function was acute and progressed faster than changes in serum bilirubin concentrations. These findings suggest that longitudinal quantitative in vivo imaging may be a useful addition to standard biological assays to gain additional knowledge about organ pathophysiology in animal models of EVD. IMPORTANCE Severe liver impairment is a well-known hallmark of Ebola virus disease (EVD), but the contribution of hepatic pathophysiology to EVD progression is not fully understood. Noninvasive medical imaging of liver structure and function in well-established animal models of disease may shed light on this important aspect of EVD. In this proof-of-concept study, we used longitudinal magnetic resonance imaging (MRI) to characterize liver abnormalities and dysfunction in rhesus monkeys exposed to Ebola virus. The results indicate that in vivo MRI may be used as a noninvasive readout of organ pathophysiology in EVD and may be used in future animal studies to further characterize organ-specific damage of this condition, in addition to standard biological assays.

Longitudinal in vivo imaging of acute neuropathology in a monkey model of Ebola virus infection.

Ebola virus (EBOV) causes neurological symptoms yet its effects on the central nervous system (CNS) are not well-described. Here, we longitudinally assess the acute effects of EBOV on the brain, using quantitative MR-relaxometry, 18F-Fluorodeoxyglucose PET and immunohistochemistry in a monkey model. We report blood-brain barrier disruption, likely related to high cytokine levels and endothelial viral infection, with extravasation of fluid, Gadolinium-based contrast material and albumin into the extracellular space. Increased glucose metabolism is also present compared to the baseline, especially in the deep gray matter and brainstem. This regional hypermetabolism corresponds with mild neuroinflammation, sporadic neuronal infection and apoptosis, as well as increased GLUT3 expression, consistent with increased neuronal metabolic demands. Neuroimaging changes are associated with markers of disease progression including viral load and cytokine/chemokine levels. Our results provide insight into the pathophysiology of CNS involvement with EBOV and may help assess vaccine/treatment efficacy in real time.

Acute Late-Stage Myocarditis in the Crab-Eating Macaque Model of Hemorrhagic Smallpox.

Hemorrhagic smallpox, caused by variola virus (VARV), was a rare but nearly 100% lethal human disease manifestation. Hemorrhagic smallpox is frequently characterized by secondary bacterial infection, coagulopathy, and myocardial and subendocardial hemorrhages. Previous experiments have demonstrated that intravenous (IV) cowpox virus (CPXV) exposure of macaques mimics human hemorrhagic smallpox. The goal of this experiment was to further understand the onset, nature, and severity of cardiac pathology and how it may contribute to disease. The findings support an acute late-stage myocarditis with lymphohistiocytic infiltrates in the CPXV model of hemorrhagic smallpox.

Clinical Characterization of Host Response to Simian Hemorrhagic Fever Virus Infection in Permissive and Refractory Hosts: A Model for Determining Mechanisms of VHF Pathogenesis.

Simian hemorrhagic fever virus (SHFV) causes a fulminant and typically lethal viral hemorrhagic fever (VHF) in macaques (Cercopithecinae: Macaca spp.) but causes subclinical infections in patas monkeys (Cercopithecinae: Erythrocebus patas). This difference in disease course offers a unique opportunity to compare host responses to infection by a VHF-causing virus in biologically similar susceptible and refractory animals. Patas and rhesus monkeys were inoculated side-by-side with SHFV. Unlike the severe disease observed in rhesus monkeys, patas monkeys developed a limited clinical disease characterized by changes in complete blood counts, serum chemistries, and development of lymphadenopathy. Viral RNA was measurable in circulating blood 2 days after exposure, and its duration varied by species. Infectious virus was detected in terminal tissues of both patas and rhesus monkeys. Varying degrees of overlap in changes in serum concentrations of interferon (IFN)-γ, monocyte chemoattractant protein (MCP)-1, and interleukin (IL)-6 were observed between patas and rhesus monkeys, suggesting the presence of common and species-specific cytokine responses to infection. Similarly, quantitative immunohistochemistry of livers from terminal monkeys and whole blood flow cytometry revealed varying degrees of overlap in changes in macrophages, natural killer cells, and T-cells. The unexpected degree of overlap in host response suggests that relatively small subsets of a host's response to infection may be responsible for driving hemorrhagic fever pathogenesis. Furthermore, comparative SHFV infection in patas and rhesus monkeys offers an experimental model to characterize host⁻response mechanisms associated with viral hemorrhagic fever and evaluate pan-viral hemorrhagic fever countermeasures.

Non-neutralizing antibodies elicited by recombinant Lassa-Rabies vaccine are critical for protection against Lassa fever.

Lassa fever (LF), caused by Lassa virus (LASV), is a viral hemorrhagic fever for which no approved vaccine or potent antiviral treatment is available. LF is a WHO priority disease and, together with rabies, a major health burden in West Africa. Here we present the development and characterization of an inactivated recombinant LASV and rabies vaccine candidate (LASSARAB) that expresses a codon-optimized LASV glycoprotein (coGPC) and is adjuvanted by a TLR-4 agonist (GLA-SE). LASSARAB elicits lasting humoral response against LASV and RABV in both mouse and guinea pig models, and it protects both guinea pigs and mice against LF. We also demonstrate a previously unexplored role for non-neutralizing LASV GPC-specific antibodies as a major mechanism of protection by LASSARAB against LF through antibody-dependent cellular functions. Overall, these findings demonstrate an effective inactivated LF vaccine and elucidate a novel humoral correlate of protection for LF.

The Human Sodium Iodide Symporter as a Reporter Gene for Studying Middle East Respiratory Syndrome Coronavirus Pathogenesis.

Single photon emission computed tomography (SPECT) is frequently used in oncology and cardiology to evaluate disease progression and/or treatment efficacy. Such technology allows for real-time evaluation of disease progression and when applied to studying infectious diseases may provide insight into pathogenesis. Insertion of a SPECT-compatible reporter gene into a virus may provide insight into mechanisms of pathogenesis and viral tropism. The human sodium iodide symporter (hNIS), a SPECT and positron emission tomography reporter gene, was inserted into Middle East respiratory syndrome coronavirus (MERS-CoV), a recently emerged virus that can cause severe respiratory disease and death in afflicted humans to obtain a quantifiable and sensitive marker for viral replication to further MERS-CoV animal model development. The recombinant virus was evaluated for fitness, stability, and reporter gene functionality. The recombinant and parental viruses demonstrated equal fitness in terms of peak titer and replication kinetics, were stable for up to six in vitro passages, and were functional. Further in vivo evaluation indicated variable stability, but resolution limits hampered in vivo functional evaluation. These data support the further development of hNIS for monitoring infection in animal models of viral disease.IMPORTANCE Advanced medical imaging such as single photon emission computed tomography with computed tomography (SPECT/CT) enhances fields such as oncology and cardiology. Application of SPECT/CT, magnetic resonance imaging, and positron emission tomography to infectious disease may enhance pathogenesis studies and provide alternate biomarkers of disease progression. The experiments described in this article focus on insertion of a SPECT/CT-compatible reporter gene into MERS-CoV to demonstrate that a functional SPECT/CT reporter gene can be inserted into a virus.

Silencing CDK4 radiosensitizes breast cancer cells by promoting apoptosis.

BACKGROUND: The discovery of molecular markers associated with various breast cancer subtypes has greatly improved the treatment and outcome of breast cancer patients. Unfortunately, breast cancer cells acquire resistance to various therapies. Mounting evidence suggests that resistance is rooted in the deregulation of the G1 phase regulatory machinery. METHODS: To address whether deregulation of the G1 phase regulatory machinery contributes to radiotherapy resistance, the MCF10A immortalized human mammary epithelial cell line, ER-PR-Her2+ and ER-PR-Her2- breast cancer cell lines were irradiated. Colony formation assays measured radioresistance, while immunocytochemistry, Western blots, and flow cytometry measured the cell cycle, DNA replication, mitosis, apoptosis, and DNA breaks. RESULTS: Molecular markers common to all cell lines were overexpressed, including cyclin A1 and cyclin D1, which impinge on CDK2 and CDK4 activities, respectively. We addressed their potential role in radioresistance by generating cell lines stably expressing small hairpin RNAs (shRNA) against CDK2 and CDK4. None of the cell lines knocked down for CDK2 displayed radiosensitization. In contrast, all cell lines knocked down for CDK4 were significantly radiosensitized, and a CDK4/CDK6 inhibitor sensitized MDA-MB-468 to radiation induced apoptosis. Our data showed that silencing CDK4 significantly increases radiation induced cell apoptosis in cell lines without significantly altering cell cycle progression, or DNA repair after irradiation. Our results indicate lower levels of phospho-Bad at ser136 upon CDK4 silencing and ionizing radiation, which has been shown to signal apoptosis. CONCLUSION: Based on our data we conclude that knockdown of CDK4 activity sensitizes breast cancer cells to radiation by activating apoptosis pathways.