Hepatitis C Virus Antiviral Drug Resistance and Salvage Therapy Outcomes Across Australia.

Background: Hepatitis C virus (HCV) infection can now be cured with well-tolerated direct-acting antiviral (DAA) therapy. However, a potential barrier to HCV elimination is the emergence of resistance-associated substitutions (RASs) that reduce the efficacy of antiviral drugs, but real-world studies assessing the clinical impact of RASs are limited. Here, an analysis of the impact of RASs on retreatment outcomes for different salvage regimens in patients nationally who failed first-line DAA therapy is reported. Methods: We collected data from 363 Australian patients who failed first-line DAA therapy, including: age, sex, fibrosis stage, HCV genotype, NS3/NS5A/NS5B RASs, details of failed first-line regimen, subsequent salvage regimens, and treatment outcome. Results: Of 240 patients who were initially retreated as per protocol, 210 (87.5%) achieved sustained virologic response (SVR) and 30 (12.5%) relapsed or did not respond. The SVR rate for salvage regimens that included sofosbuvir/velpatasvir/voxilaprevir was 94.3% (n = 140), sofosbuvir/velpatasvir 75.0% (n = 52), elbasvir/grazoprevir 81.6% (n = 38), and glecaprevir/pibrentasvir 84.6% (n = 13). NS5A RASs were present in 71.0% (n = 210) of patients who achieved SVR and in 66.7% (n = 30) of patients who subsequently relapsed. NS3 RASs were detected in 20 patients (20%) in the SVR group and 1 patient in the relapse group. NS5B RASs were observed in only 3 patients. Cirrhosis was a predictor of relapse after retreatment, as was previous treatment with sofosbuvir/velpatasvir. Conclusions: In our cohort, the SVR rate for sofosbuvir/velpatasvir/voxilaprevir was higher than with other salvage regimens. The presence of NS5A, NS5B, or NS3 RASs did not appear to negatively influence retreatment outcomes.

Dynamic cycling with a unique Hsp90/Hsp70-dependent chaperone machinery and GAPDH is needed for heme insertion and activation of neuronal NO synthase.

Heat shock protein 90 (Hsp90) is known to mediate heme insertion and activation of heme-deficient neuronal nitric oxide (NO) synthase (apo-nNOS) in cells by a highly dynamic interaction that has been extremely difficult to study mechanistically with the use of subcellular systems. In that the heme content of many critical hemeproteins is regulated by Hsp90 and the heme chaperone GAPDH, the development of an in vitro system for the study of this chaperone-mediated heme regulation would be extremely useful. Here, we show that use of an antibody-immobilized apo-nNOS led not only to successful assembly of chaperone complexes but the ability to show a clear dependence on Hsp90 and GAPDH for heme-mediated activation of apo-nNOS. The kinetics of binding for Hsp70 and Hsp90, the ATP and K+ dependence, and the absolute requirement for Hsp70 in assembly of Hsp90•apo-nNOS heterocomplexes all point to a similar chaperone machinery to the well-established canonical machine regulating steroid hormone receptors. However, unlike steroid receptors, the use of a purified protein system containing Hsp90, Hsp70, Hsp40, Hop, and p23 is unable to activate apo-nNOS. Thus, heme insertion requires a unique Hsp90-chaperone complex. With this newly developed in vitro system, which recapitulates the cellular process requiring GAPDH as well as Hsp90, further mechanistic studies are now possible to better understand the components of the Hsp90-based chaperone system as well as how this heterocomplex works with GAPDH to regulate nNOS and possibly other hemeproteins.

Natural History of Aerosol-Induced Ebola Virus Disease in Rhesus Macaques.

Ebola virus disease (EVD) is a serious global health concern because case fatality rates are approximately 50% due to recent widespread outbreaks in Africa. Well-defined nonhuman primate (NHP) models for different routes of Ebola virus exposure are needed to test the efficacy of candidate countermeasures. In this natural history study, four rhesus macaques were challenged via aerosol with a target titer of 1000 plaque-forming units per milliliter of Ebola virus. The course of disease was split into the following stages for descriptive purposes: subclinical, clinical, and decompensated. During the subclinical stage, high levels of venous partial pressure of carbon dioxide led to respiratory acidemia in three of four of the NHPs, and all developed lymphopenia. During the clinical stage, all animals had fever, viremia, and respiratory alkalosis. The decompensatory stage involved coagulopathy, cytokine storm, and liver and renal injury. These events were followed by hypotension, elevated lactate, metabolic acidemia, shock and mortality similar to historic intramuscular challenge studies. Viral loads in the lungs of aerosol-exposed animals were not distinctly different compared to previous intramuscularly challenged studies. Differences in the aerosol model, compared to intramuscular model, include an extended subclinical stage, shortened clinical stage, and general decompensated stage. Therefore, the shortened timeframe for clinical detection of the aerosol-induced disease can impair timely therapeutic administration. In summary, this nonhuman primate model of aerosol-induced EVD characterizes early disease markers and additional details to enable countermeasure development.

Detecting Pathogen Exposure During the Non-symptomatic Incubation Period Using Physiological Data: Proof of Concept in Non-human Primates.

Background and Objectives: Early warning of bacterial and viral infection, prior to the development of overt clinical symptoms, allows not only for improved patient care and outcomes but also enables faster implementation of public health measures (patient isolation and contact tracing). Our primary objectives in this effort are 3-fold. First, we seek to determine the upper limits of early warning detection through physiological measurements. Second, we investigate whether the detected physiological response is specific to the pathogen. Third, we explore the feasibility of extending early warning detection with wearable devices. Research Methods: For the first objective, we developed a supervised random forest algorithm to detect pathogen exposure in the asymptomatic period prior to overt symptoms (fever). We used high-resolution physiological telemetry data (aortic blood pressure, intrathoracic pressure, electrocardiograms, and core temperature) from non-human primate animal models exposed to two viral pathogens: Ebola and Marburg (N = 20). Second, to determine reusability across different pathogens, we evaluated our algorithm against three independent physiological datasets from non-human primate models (N = 13) exposed to three different pathogens: Lassa and Nipah viruses and Y. pestis. For the third objective, we evaluated performance degradation when the algorithm was restricted to features derived from electrocardiogram (ECG) waveforms to emulate data from a non-invasive wearable device. Results: First, our cross-validated random forest classifier provides a mean early warning of 51 ± 12 h, with an area under the receiver-operating characteristic curve (AUC) of 0.93 ± 0.01. Second, our algorithm achieved comparable performance when applied to datasets from different pathogen exposures - a mean early warning of 51 ± 14 h and AUC of 0.95 ± 0.01. Last, with a degraded feature set derived solely from ECG, we observed minimal degradation - a mean early warning of 46 ± 14 h and AUC of 0.91 ± 0.001. Conclusion: Under controlled experimental conditions, physiological measurements can provide over 2 days of early warning with high AUC. Deviations in physiological signals following exposure to a pathogen are due to the underlying host's immunological response and are not specific to the pathogen. Pre-symptomatic detection is strong even when features are limited to ECG-derivatives, suggesting that this approach may translate to non-invasive wearable devices.

The utilization of advance telemetry to investigate critical physiological parameters including electroencephalography in cynomolgus macaques following aerosol challenge with eastern equine encephalitis virus.

Most alphaviruses are mosquito-borne and can cause severe disease in humans and domesticated animals. In North America, eastern equine encephalitis virus (EEEV) is an important human pathogen with case fatality rates of 30-90%. Currently, there are no therapeutics or vaccines to treat and/or prevent human infection. One critical impediment in countermeasure development is the lack of insight into clinically relevant parameters in a susceptible animal model. This study examined the disease course of EEEV in a cynomolgus macaque model utilizing advanced telemetry technology to continuously and simultaneously measure temperature, respiration, activity, heart rate, blood pressure, electrocardiogram (ECG), and electroencephalography (EEG) following an aerosol challenge at 7.0 log10 PFU. Following challenge, all parameters were rapidly and substantially altered with peak alterations from baseline ranged as follows: temperature (+3.0-4.2°C), respiration rate (+56-128%), activity (-15-76% daytime and +5-22% nighttime), heart rate (+67-190%), systolic (+44-67%) and diastolic blood pressure (+45-80%). Cardiac abnormalities comprised of alterations in QRS and PR duration, QTc Bazett, T wave morphology, amplitude of the QRS complex, and sinoatrial arrest. An unexpected finding of the study was the first documented evidence of a critical cardiac event as an immediate cause of euthanasia in one NHP. All brain waves were rapidly (~12-24 hpi) and profoundly altered with increases of up to 6,800% and severe diffuse slowing of all waves with decreases of ~99%. Lastly, all NHPs exhibited disruption of the circadian rhythm, sleep, and food/fluid intake. Accordingly, all NHPs met the euthanasia criteria by ~106-140 hpi. This is the first of its kind study utilizing state of the art telemetry to investigate multiple clinical parameters relevant to human EEEV infection in a susceptible cynomolgus macaque model. The study provides critical insights into EEEV pathogenesis and the parameters identified will improve animal model development to facilitate rapid evaluation of vaccines and therapeutics.

Intensive Care Unit-Like Care of Nonhuman Primates with Ebola Virus Disease.

BACKGROUND: Ebola virus disease (EVD) supportive care strategies are largely guided by retrospective observational research. This study investigated the effect of EVD supportive care algorithms on duration of survival in a controlled nonhuman primate (NHP) model. METHODS: Fourteen rhesus macaques were challenged intramuscularly with a target dose of Ebola virus (1000 plaque-forming units; Kikwit). NHPs were allocated to intensive care unit (ICU)-like algorithms (n = 7), intravenous fluids plus levofloxacin (n = 2), or a control group (n = 5). The primary outcome measure was duration of survival, and secondary outcomes included changes in clinical laboratory values. RESULTS: Duration of survival was not significantly different between the pooled ICU-like algorithm and control groups (8.2 vs 6.9 days of survival; hazard ratio; 0.50; P = .25). Norepinephrine was effective in transiently maintaining baseline blood pressure. NHPs treated with ICU-like algorithms had delayed onset of liver and kidney injury. CONCLUSIONS: While an obvious survival difference was not observed with ICU-like care, clinical observations from this model may aid in EVD supportive care NHP model refinement.

Translation of evidence into policy to improve clinical practice: the development of an emergency department rapid response system.

BACKGROUND: Undetected clinical deterioration is a major cause of high mortality events in Emergency Department (ED) patients. Yet, there is no known model to guide the recognition and response to clinical deterioration in the ED, integrating internal and external resources. METHODS: An integrative review was firstly conducted to identify the critical components of recognising and responding to clinical deterioration in the ED. Components identified from the review were analysed by clinical experts and informed the development of an ED Clinical Emergency Response System (EDCERS). RESULTS: Twenty four eligible studies were included in the review. Eight core components were identified: 1) vital sign monitoring; 2) track and trigger system; 3) communication plan; 4) response time; 5) emergency nurse response; 6) emergency physician response; 7) critical care team response; and 8) specialty team response. These components informed the development of the EDCERS protocol, integrating responses from staff internal and external to the ED. CONCLUSIONS: EDCERS was based on the best available evidence and considered the cultural context of care. Future research is needed to determine the useability and impact of EDCERS on patient and health outcomes.

Low-Dose Hsp90 Inhibitor Selectively Radiosensitizes HNSCC and Pancreatic Xenografts.

PURPOSE: Treatment approaches using Hsp90 inhibitors at their maximum tolerated doses (MTDs) have not produced selective tumor toxicity. Inhibition of Hsp90 activity causes degradation of client proteins including those involved in recognizing and repairing DNA lesions. We hypothesized that if DNA repair proteins were degraded by concentrations of an Hsp90 inhibitor below those required to cause nonspecific cytotoxicity, significant tumor-selective radiosensitization might be achieved. EXPERIMENTAL DESIGN: Tandem mass tagged-mass spectrometry was performed to determine the effect of a subcytotoxic concentration of the Hsp90 inhibitor, AT13387 (onalespib), on global protein abundance. The effect of AT13387 on in vitro radiosensitization was assessed using a clonogenic assay. Pharmacokinetics profiling was performed in mice bearing xenografts. Finally, the effect of low-dose AT13387 on the radiosensitization of three tumor models was assessed. RESULTS: A subcytotoxic concentration of AT13387 reduced levels of DNA repair proteins, without affecting the majority of Hsp90 clients. The pharmacokinetics study using one-third of the MTD showed 40-fold higher levels of AT13387 in tumors compared with plasma. This low dose enhanced Hsp70 expression in peripheral blood mononuclear cells (PBMCs), which is a biomarker of Hsp90 inhibition. Low dose monotherapy was ineffective, but when combined with radiotherapy, produced significant tumor growth inhibition. CONCLUSIONS: This study shows that a significant therapeutic ratio can be achieved by a low dose of Hsp90 inhibitor in combination with radiotherapy. Hsp90 inhibition, even at a low dose, can be monitored by measuring Hsp70 expression in PBMCs in human studies.

Natural History of Aerosol Induced Lassa Fever in Non­Human Primates.

Lassa virus (LASV), an arenavirus causing Lassa fever, is endemic to West Africa with up to 300,000 cases and between 5000 and 10,000 deaths per year. Rarely seen in the United States, Lassa virus is a CDC category A biological agent inasmuch deliberate aerosol exposure can have high mortality rates compared to naturally acquired infection. With the need for an animal model, specific countermeasures remain elusive as there is no FDA-approved vaccine. This natural history of aerosolized Lassa virus exposure in Macaca fascicularis was studied under continuous telemetric surveillance. The macaque response to challenge was largely analogous to severe human disease with fever, tachycardia, hypotension, and tachypnea. During initial observations, an increase trend of activated monocytes positive for viral glycoprotein was accompanied by lymphocytopenia. Disease uniformly progressed to high viremia followed by low anion gap, alkalosis, anemia, and thrombocytopenia. Hypoproteinemia occurred late in infection followed by increased levels of white blood cells, cytokines, chemokines, and biochemical markers of liver injury. Viral nucleic acids were detected in tissues of three non­survivors at endpoint, but not in the lone survivor. This study provides useful details to benchmark a pivotal model of Lassa fever in support of medical countermeasure development for both endemic disease and traditional biodefense purposes.

Modeling mosquito-borne and sexual transmission of Zika virus in an enzootic host, the African green monkey.

Mosquito-borne and sexual transmission of Zika virus (ZIKV), a TORCH pathogen, recently initiated a series of large epidemics throughout the Tropics. Animal models are necessary to determine transmission risk and study pathogenesis, as well screen antivirals and vaccine candidates. In this study, we modeled mosquito and sexual transmission of ZIKV in the African green monkey (AGM). Following subcutaneous, intravaginal or intrarectal inoculation of AGMs with ZIKV, we determined the transmission potential and infection dynamics of the virus. AGMs inoculated by all three transmission routes exhibited viremia and viral shedding followed by strong virus neutralizing antibody responses, in the absence of clinical illness. All four of the subcutaneously inoculated AGMs became infected (mean peak viremia: 2.9 log10 PFU/mL, mean duration: 4.3 days) and vRNA was detected in their oral swabs, with infectious virus being detected in a subset of these specimens. Although all four of the intravaginally inoculated AGMs developed virus neutralizing antibody responses, only three had detectable viremia (mean peak viremia: 4.0 log10 PFU/mL, mean duration: 3.0 days). These three AGMs also had vRNA and infectious virus detected in both oral and vaginal swabs. Two of the four intrarectally inoculated AGMs became infected (mean peak viremia: 3.8 log10 PFU/mL, mean duration: 3.5 days). vRNA was detected in oral swabs collected from both of these infected AGMs, and infectious virus was detected in an oral swab from one of these AGMs. Notably, vRNA and infectious virus were detected in vaginal swabs collected from the infected female AGM (peak viral load: 7.5 log10 copies/mL, peak titer: 3.8 log10 PFU/mL, range of detection: 5-21 days post infection). Abnormal clinical chemistry and hematology results were detected and acute lymphadenopathy was observed in some AGMs. Infection dynamics in all three AGM ZIKV models are similar to those reported in the majority of human ZIKV infections. Our results indicate that the AGM can be used as a surrogate to model mosquito or sexual ZIKV transmission and infection. Furthermore, our results suggest that AGMs are likely involved in the enzootic maintenance and amplification cycle of ZIKV.

Targeting Hsp70 facilitated protein quality control for treatment of polyglutamine diseases.

The polyglutamine (polyQ) diseases are a group of nine fatal, adult-onset neurodegenerative disorders characterized by the misfolding and aggregation of mutant proteins containing toxic expansions of CAG/polyQ tracts. The heat shock protein 90 and 70 (Hsp90/Hsp70) chaperone machinery is a key component of cellular protein quality control, playing a role in the regulation of folding, aggregation, and degradation of polyQ proteins. The ability of Hsp70 to facilitate disaggregation and degradation of misfolded proteins makes it an attractive therapeutic target in polyQ diseases. Genetic studies have demonstrated that manipulation of Hsp70 and related co-chaperones can enhance the disaggregation and/or degradation of misfolded proteins in models of polyQ disease. Therefore, the development of small molecules that enhance Hsp70 activity is of great interest. However, it is still unclear if currently available Hsp70 modulators can selectively enhance disaggregation or degradation of misfolded proteins without perturbing other Hsp70 functions essential for cellular homeostasis. This review discusses the multifaceted role of Hsp70 in protein quality control and the opportunities and challenges Hsp70 poses as a potential therapeutic target in polyQ disease.

Persistent Crimean-Congo hemorrhagic fever virus infection in the testes and within granulomas of non-human primates with latent tuberculosis.

Crimean-Congo hemorrhagic fever (CCHF) is the most medically important tick-borne viral disease of humans and tuberculosis is the leading cause of death worldwide by a bacterial pathogen. These two diseases overlap geographically, however, concurrent infection of CCHF virus (CCHFV) with mycobacterial infection has not been assessed nor has the ability of virus to persist and cause long-term sequela in a primate model. In this study, we compared the disease progression of two diverse strains of CCHFV in the recently described cynomolgus macaque model. All animals demonstrated signs of clinical illness, viremia, significant changes in clinical chemistry and hematology values, and serum cytokine profiles consistent with CCHF in humans. The European and Asian CCHFV strains caused very similar disease profiles in monkeys, which demonstrates that medical countermeasures can be evaluated in this animal model against multiple CCHFV strains. We identified evidence of CCHFV persistence in the testes of three male monkeys that survived infection. Furthermore, the histopathology unexpectedly revealed that six additional animals had evidence of a latent mycobacterial infection with granulomatous lesions. Interestingly, CCHFV persisted within the granulomas of two animals. This study is the first to demonstrate the persistence of CCHFV in the testes and within the granulomas of non-human primates with concurrent latent tuberculosis. Our results have important public health implications in overlapping endemic regions for these emerging pathogens.

Phase control in a spin-triplet SQUID.

It is now well established that a Josephson junction made from conventional spin-singlet superconductors containing ferromagnetic layers can carry spin-triplet supercurrent under certain conditions. The first experimental signature of that fact is the propagation of such supercurrent over long distances through strong ferromagnetic materials. Surprisingly, one of the most salient predictions of the theory has yet to be verified experimentally-namely, that a Josephson junction containing three magnetic layers with coplanar magnetizations should exhibit a ground-state phase shift of either zero or π depending on the relative orientations of those magnetizations. We demonstrate this property using Josephson junctions containing three different types of magnetic layers, chosen so that the magnetization of one layer can be switched by 180° without disturbing the other two. Phase-sensitive detection is accomplished using a superconducting quantum interference device, or SQUID. Such a phase-controllable junction could be used as the memory element in a fully superconducting computer.

Chaperone Activity and Dimerization Properties of Hsp90α and Hsp90ß in Glucocorticoid Receptor Activation by the Multiprotein Hsp90/Hsp70-Dependent Chaperone Machinery.

Several hundred proteins cycle into heterocomplexes with a dimer of the chaperone heat shock protein 90 (Hsp90), regulating their activity and turnover. There are two isoforms of Hsp90, Hsp90α and Hsp90ß, and their relative chaperone activities and composition in these client protein•Hsp90 heterocomplexes has not been determined. Here, we examined the activity of human Hsp90α and Hsp90ß in a purified five-protein chaperone machinery that assembles glucocorticoid receptor (GR)•Hsp90 heterocomplexes to generate high-affinity steroid-binding activity. We found that human Hsp90α and Hsp90ß have equivalent chaperone activities, and when mixed together in this assay, they formed only GR•Hsp90αα and GR•Hsp90ßß homodimers and no GR•Hsp90αß heterodimers. In contrast, GR•Hsp90 heterocomplexes formed in human embryonic kidney (HEK) cells also contain GR•Hsp90αß heterodimers. The formation of GR•Hsp90αß heterodimers in HEK cells probably reflects the longer time permitted for exchange to form Hsp90αß heterodimers in the cell versus in the cell-free assembly conditions. This purified GR-activating chaperone machinery can be used to determine how modifications of Hsp90 affect its chaperone activity. To that effect, we have tested whether the unique phosphorylation of Hsp90α at threonines 5 and 7 that occurs during DNA damage repair affects its chaperone activity. We showed that the phosphomimetic mutant Hsp90α T5/7D has the same intrinsic chaperone activity as wild-type human Hsp90α in activation of GR steroid-binding activity by the five-protein machinery, supporting the conclusion that T5/7 phosphorylation does not affect Hsp90α chaperone activity.

Development of Clinical-Stage Human Monoclonal Antibodies That Treat Advanced Ebola Virus Disease in Nonhuman Primates.

Background: For most classes of drugs, rapid development of therapeutics to treat emerging infections is challenged by the timelines needed to identify compounds with the desired efficacy, safety, and pharmacokinetic profiles. Fully human monoclonal antibodies (mAbs) provide an attractive method to overcome many of these hurdles to rapidly produce therapeutics for emerging diseases. Methods: In this study, we deployed a platform to generate, test, and develop fully human antibodies to Zaire ebolavirus. We obtained specific anti-Ebola virus (EBOV) antibodies by immunizing VelocImmune mice that use human immunoglobulin variable regions in their humoral responses. Results: Of the antibody clones isolated, 3 were selected as best at neutralizing EBOV and triggering FcγRIIIa. Binding studies and negative-stain electron microscopy revealed that the 3 selected antibodies bind to non-overlapping epitopes, including a potentially new protective epitope not targeted by other antibody-based treatments. When combined, a single dose of a cocktail of the 3 antibodies protected nonhuman primates (NHPs) from EBOV disease even after disease symptoms were apparent. Conclusions: This antibody cocktail provides complementary mechanisms of actions, incorporates novel specificities, and demonstrates high-level postexposure protection from lethal EBOV disease in NHPs. It is now undergoing testing in normal healthy volunteers in preparation for potential use in future Ebola epidemics.

A conserved transcriptional response to intranasal Ebola virus exposure in nonhuman primates prior to onset of fever.

Ebola virus disease (EVD), caused by Ebola virus (EBOV), is a severe illness characterized by case fatality rates of up to 90%. The sporadic nature of outbreaks in resource-limited areas has hindered the ability to characterize the pathogenesis of EVD at all stages of infection but particularly early host responses. Pathogenesis is often studied in nonhuman primate (NHP) models of disease that replicate major aspects of human EVD. Typically, NHP models use a large infectious dose, are carried out through intramuscular or aerosol exposure, and have a fairly uniform disease course. By contrast, we report our analysis of the host response to EBOV after intranasal exposure. Twelve cynomolgus macaques were infected with 100 plaque-forming units of EBOV/Makona through intranasal exposure and presented with varying times to onset of EVD. We used RNA sequencing and a newly developed NanoString CodeSet to monitor the host response via changes in RNA transcripts over time. When individual animal gene expression data were phased based on the onset of sustained fever, the first clinical sign of severe disease, mathematical models indicated that interferon-stimulated genes appeared as early as 4 days before fever onset. This demonstrates that lethal EVD has a uniform and predictable response to infection regardless of time to onset. Furthermore, expression of a subset of genes could predict disease development before other host-based indications of infection such as fever.

Recombinant proteins of Zaire ebolavirus induce potent humoral and cellular immune responses and protect against live virus infection in mice.

Infections with filoviruses in humans are highly virulent, causing hemorrhagic fevers which result in up to 90% mortality. In addition to natural infections, the ability to use these viruses as bioterrorist weapons is of significant concern. Currently, there are no licensed vaccines or therapeutics available to combat these infections. The pathogenesis of disease involves the dysregulation of the host's immune system, which results in impairment of the innate and adaptive immune responses, with subsequent development of lymphopenia, thrombocytopenia, hemorrhage, and death. Questions remain with regard to the few survivors of infection, who manage to mount an effective adaptive immune response. These questions concern the humoral and cellular components of this response, and whether such a response can be elicited by an appropriate prophylactic vaccine. The data reported herein describe the production and evaluation of a recombinant subunit Ebola virus vaccine candidate consisting of insect cell expressed Zaire ebolavirus (EBOV) surface glycoprotein (GP) and the matrix proteins VP24 and VP40. The recombinant subunit proteins are shown to be highly immunogenic in mice, yielding both humoral and cellular responses, as well as highly efficacious, providing up to 100% protection against a lethal challenge with live virus. These results demonstrate proof of concept for such a recombinant non-replicating vaccine candidate in the mouse model of EBOV which helps to elucidate immune correlates of protection and warrants further development.

High Infection Rates for Adult Macaques after Intravaginal or Intrarectal Inoculation with Zika Virus.

Unprotected sexual intercourse between persons residing in or traveling from regions with Zika virus transmission is a risk factor for infection. To model risk for infection after sexual intercourse, we inoculated rhesus and cynomolgus macaques with Zika virus by intravaginal or intrarectal routes. In macaques inoculated intravaginally, we detected viremia and virus RNA in 50% of macaques, followed by seroconversion. In macaques inoculated intrarectally, we detected viremia, virus RNA, or both, in 100% of both species, followed by seroconversion. The magnitude and duration of infectious virus in the blood of macaques suggest humans infected with Zika virus through sexual transmission will likely generate viremias sufficient to infect competent mosquito vectors. Our results indicate that transmission of Zika virus by sexual intercourse might serve as a virus maintenance mechanism in the absence of mosquito-to-human transmission and could increase the probability of establishment and spread of Zika virus in regions where this virus is not present.

ATM is the primary kinase responsible for phosphorylation of Hsp90α after ionizing radiation.

Heat shock protein 90 is a chaperone that plays an essential role in the stabilization of a large number of signal transduction molecules, many of which are associated with oncogenesis. An Hsp90 isoform (Hsp90α) has been shown to be selectively phosphorylated on two N-terminal threonine residues (threonine 5 and 7) and is involved in the DNA damage response and apoptosis. However, the kinase that phosphorylates Hsp90α after ionizing radiation (IR) and its role in post-radiation DNA repair remains unclear. Inasmuch as several proteins of the DNA damage response machinery are Hsp90 clients, the functional consequences of Hsp90α phosphorylation following IR have implications for the design of novel radiosensitizing agents that specifically target the Hsp90α isoform. Here we show that ATM phosphorylates Hsp90α at the T5/7 residues immediately after IR. The kinetics of Hsp90α T5/7 phosphorylation correlate with the kinetics of H2AX S139 phosphorylation (γH2AX). Although Hsp90α is located in both the cytoplasm and nucleus, only nuclear Hsp90α is phosphorylated by ATM after IR. The siRNA mediated knockdown of Hsp90α sensitizes head and neck squamous cell carcinoma cells, lung cancer cells and lung fibroblasts to IR. Furthermore, MEF cells that are Hsp90α null have reduced levels of γH2AX indicating that Hsp90α is important for the formation of γH2AX. Thus, this study provides evidence that Hsp90α is a component of the signal transduction events mediated by ATM following IR, and that Hsp90α loss decreases γH2AX levels. This work supports additional investigation into Hsp90α T5/7 phosphorylation with the goal of developing targeted radiosensitizing therapies.

Natural History of Aerosol Exposure with Marburg Virus in Rhesus Macaques.

Marburg virus causes severe and often lethal viral disease in humans, and there are currently no Food and Drug Administration (FDA) approved medical countermeasures. The sporadic occurrence of Marburg outbreaks does not allow for evaluation of countermeasures in humans, so therapeutic and vaccine candidates can only be approved through the FDA animal rule-a mechanism requiring well-characterized animal models in which efficacy would be evaluated. Here, we describe a natural history study where rhesus macaques were surgically implanted with telemetry devices and central venous catheters prior to aerosol exposure with Marburg-Angola virus, enabling continuous physiologic monitoring and blood sampling without anesthesia. After a three to four day incubation period, all animals developed fever, viremia, and lymphopenia before developing tachycardia, tachypnea, elevated liver enzymes, decreased liver function, azotemia, elevated D-dimer levels and elevated pro-inflammatory cytokines suggesting a systemic inflammatory response with organ failure. The final, terminal period began with the onset of sustained hypotension, dehydration progressed with signs of major organ hypoperfusion (hyperlactatemia, acute kidney injury, hypothermia), and ended with euthanasia or death. The most significant pathologic findings were marked infection of the respiratory lymphoid tissue with destruction of the tracheobronchial and mediastinal lymph nodes, and severe diffuse infection in the liver, and splenitis.