Search

A 46-year-old patient with previously documented Ebola virus persistence in his ocular fluid, associated with severe panuveitis, developed a visually significant cataract. A multidisciplinary approach was taken to prevent and control infection. Ebola virus persistence was assessed before and during the operation to provide safe, vision-restorative phacoemulsification surgery.

Marburg virus (MARV) is a filovirus with documented human case-fatality rates of up to 90%. Here, we evaluated the therapeutic efficacy of remdesivir (GS-5734) in nonhuman primates experimentally infected with MARV. Beginning 4 or 5 days post inoculation, cynomolgus macaques were treated once daily for 12 days with vehicle, 5 mg/kg remdesivir, or a 10-mg/kg loading dose followed by 5 mg/kg remdesivir. All vehicle-control animals died, whereas 83% of animals receiving a 10-mg/kg loading dose of remdesivir survived, as did 50% of animals receiving a 5-mg/kg remdesivir regimen. Remdesivir-treated animals exhibited improved clinical scores, lower plasma viral RNA, and improved markers of kidney function, liver function, and coagulopathy versus vehicle-control animals. The small molecule remdesivir showed therapeutic efficacy in this Marburg virus disease model with treatment initiation 5 days post inoculation, supporting further assessment of remdesivir for the treatment of Marburg virus disease in humans.

A vaccine against COVID-19 is urgently needed for older adults, in whom morbidity and mortality due to the disease are increased. We aimed to assess the safety, tolerability, and immunogenicity of a candidate COVID-19 vaccine, CoronaVac, containing inactivated SARS-CoV-2, in adults aged 60 years and older.We did a randomised, double-blind, placebo-controlled, phase 1/2 clinical trial of CoronaVac in healthy adults aged 60 years and older in Renqiu (Hebei, China). Vaccine or placebo was given by intramuscular injection in two doses (days 0 and 28). Phase 1 comprised a dose-escalation study, in which participants were allocated to two blocks: block 1 (3 μg inactivated virus in 0·5 mL of aluminium hydroxide solution per injection) and block 2 (6 μg per injection). Within each block, participants were randomly assigned (2:1) using block randomisation to receive CoronaVac or placebo (aluminium hydroxide solution only). In phase 2, participants were randomly assigned (2:2:2:1) using block randomisation to receive either CoronaVac at 1·5 μg, 3 μg, or 6 μg per dose, or placebo. All participants, investigators, and laboratory staff were masked to treatment allocation. The primary safety endpoint was adverse reactions within 28 days after each injection in all participants who received at least one dose. The primary immunogenicity endpoint was seroconversion rate at 28 days after the second injection (which was assessed in all participants who had received the two doses of vaccine according to their random assignment, had antibody results available, and did not violate the trial protocol). Seroconversion was defined as a change from seronegative at baseline to seropositive for neutralising antibodies to live SARS-CoV-2 (positive cutoff titre 1/8), or a four-fold titre increase if the participant was seropositive at baseline. This study is ongoing and is registered with ClinicalTrials.gov (NCT04383574).Between May 22 and June 1, 2020, 72 participants (24 in each intervention group and 24 in the placebo group; mean age 65·8 years [SD 4·8]) were enrolled in phase 1, and between June 12 and June 15, 2020, 350 participants were enrolled in phase 2 (100 in each intervention group and 50 in the placebo group; mean age 66·6 years [SD 4·7] in 349 participants). In the safety populations from both phases, any adverse reaction within 28 days after injection occurred in 20 (20%) of 100 participants in the 1·5 μg group, 25 (20%) of 125 in the 3 μg group, 27 (22%) of 123 in the 6 μg group, and 15 (21%) of 73 in the placebo group. All adverse reactions were mild or moderate in severity and injection site pain (39 [9%] of 421 participants) was the most frequently reported event. As of Aug 28, 2020, eight serious adverse events, considered unrelated to vaccination, have been reported by seven (2%) participants. In phase 1, seroconversion after the second dose was observed in 24 of 24 participants (100·0% [95% CI 85·8-100·0]) in the 3 μg group and 22 of 23 (95·7% [78·1-99·9]) in the 6 μg group. In phase 2, seroconversion was seen in 88 of 97 participants in the 1·5 μg group (90·7% [83·1-95·7]), 96 of 98 in the 3 μg group (98·0% [92·8-99·8]), and 97 of 98 (99·0% [94·5-100·0]) in the 6 μg group. There were no detectable antibody responses in the placebo groups.CoronaVac is safe and well tolerated in older adults. Neutralising antibody titres induced by the 3 μg dose were similar to those of the 6 μg dose, and higher than those of the 1·5 μg dose, supporting the use of the 3 μg dose CoronaVac in phase 3 trials to assess protection against COVID-19.Chinese National Key Research and Development Program and Beijing Science and Technology Program.

Botulinum neurotoxins (BoNTs) are known as the most potent bacterial toxins, which can cause potentially deadly disease botulism. BoNT Serotype A (BoNT/A) is the most studied serotype as it is responsible for most human botulism cases, and its formulations are extensively utilized in clinics for therapeutic and cosmetic applications. BoNT/A has the longest-lasting effect in neurons compared to other serotypes, and there has been high interest in understanding how BoNT/A manages to escape protein degradation machinery in neurons for months. Recent work demonstrated that an E3 ligase, HECTD2, leads to efficient ubiquitination of the BoNT/A Light Chain (A/LC); however, the dominant activity of a deubiquitinase (DUB), VCIP135, inhibits the degradation of the enzymatic component. Another DUB, USP9X, was also identified as a potential indirect contributor to A/LC degradation. In this study, we screened a focused ubiquitin-proteasome pathway inhibitor library, including VCIP135 and USP9X inhibitors, and identified ten potential lead compounds affecting BoNT/A mediated SNAP-25 cleavage in neurons in pre-intoxication conditions. We then tested the dose-dependent effects of the compounds and their potential toxic effects in cells. A subset of the lead compounds demonstrated efficacy on the stability and ubiquitination of A/LC in cells. Three of the compounds, WP1130 (degrasyn), PR-619, and Celastrol, further demonstrated efficacy against BoNT/A holotoxin in an in vitro post-intoxication model. Excitingly, PR-619 and WP1130 are known inhibitors of VCIP135 and USP9X, respectively. Modulation of BoNT turnover in cells by small molecules can potentially lead to the development of effective countermeasures against botulism.

Summary Here, we show that the US Food and Drug Administration-approved oral drug nitazoxanide (NTZ) broadly amplifies the host innate immune response to viruses and inhibits Ebola virus (EBOV) replication. We find that NTZ enhances retinoic-acid-inducible protein I (RIG-I)-like-receptor, mitochondrial antiviral signaling protein, interferon regulatory factor 3, and interferon activities and induces transcription of the antiviral phosphatase GADD34. NTZ significantly inhibits EBOV replication in human cells through its effects on RIG-I and protein kinase R (PKR), suggesting that it counteracts EBOV VP35 protein's ability to block RIG-I and PKR sensing of EBOV. NTZ also inhibits a second negative-strand RNA virus, vesicular stomatitis virus (VSV), through RIG-I and GADD34, but not PKR, consistent with VSV's distinct host innate immune evasion mechanisms. Thus, NTZ counteracts varied virus-specific immune evasion strategies by generally enhancing the RNA sensing and interferon axis that is triggered by foreign cytoplasmic RNA exposure, and holds promise as an oral therapy against EBOV., Graphical Abstract, Highlights • NTZ amplifies RNA sensor and type I interferon activities and induces GADD34 expression • NTZ inhibits infectious Ebola virus (EBOV) via RIG-I and PKR, but not GADD34 • NTZ inhibits a second negative-strand RNA virus, VSV, via RIG-I and GADD34, but not PKR • NTZ holds promise as an oral therapy against EBOV, Mechanism of Action; Pathogenic Organism; Immune Response; Viral Microbiology

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., Author summary In North America, EEEV causes the most severe mosquito-borne disease in humans highlighted by fatal encephalitis and permeant debilitating neurological sequelae in survivors. The first confirmed human cases were reported more than 80 years ago and since then multiple sporadic outbreaks have occurred including one of the largest in 2019. Unfortunately, most human infections are diagnosed at the on-set of severe neurological symptoms and consequently a detailed disease course in humans is lacking. This gap in knowledge is a significant obstacle in the development of appropriate animal models to evaluate countermeasures. Here, we performed a cutting-edge study by utilizing a new telemetry technology to understand the course of EEEV infection in a susceptible macaque model by measuring multiple physiological parameters relevant to human disease. Our study demonstrates that the infection rapidly produces considerable alterations in many critical parameters including the electrical activity of the heart and the brain leading to severe disease. The study also highlights the extraordinary potential of new telemetry technology to develop the next generation of animal models to comprehensively investigate pathogenesis as well as evaluate countermeasures to treat and/or prevent EEEV disease.

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.Author SummaryIn North America, EEEV causes the most severe mosquito-borne disease in humans highlighted by fatal encephalitis and permeant debilitating neurological sequelae in survivors. The first confirmed human cases were reported more than 80 years ago and since then multiple sporadic outbreaks have occurred including one of the largest in 2019. Unfortunately, most human infections are diagnosed at the on-set of severe neurological symptoms and consequently a detailed disease course in humans is lacking. This gap in knowledge is a significant obstacle in the development of appropriate animal models to evaluate countermeasures. Here, we performed a cutting-edge study by utilizing a new telemetry technology to understand the course of EEEV infection in a susceptible macaque model by measuring multiple physiological parameters relevant to human disease. Our study demonstrates that the infection rapidly produces considerable alterations in many critical parameters including the electrical activity of the heart and the brain leading to severe disease. The study also highlights the extraordinary potential of new telemetry technology to develop the next generation of animal models in order to comprehensively investigate pathogenesis as well as evaluate countermeasures to treat and/or prevent EEEV disease.

Eastern equine encephalitis virus (EEEV) is mosquito-borne virus that produces fatal encephalitis in humans. We recently conducted a first of its kind study to investigate EEEV clinical disease course following aerosol challenge in a cynomolgus macaque model utilizing the state of the art telemetry to measure critical physiological parameters. Following challenge, all parameters were rapidly and profoundly altered, and all nonhuman primates (NHPs) met the euthanasia criteria. In this study, we performed the first comprehensive pathology investigation of tissues collected at euthanasia to gain insights into EEEV pathogenesis. Viral RNA and proteins as well as microscopic lesions were absent in the visceral organs. In contrast, viral RNA and proteins were readily detected throughout the brain including autonomic nervous system (ANS) control centers and spinal cord. However, despite presence of viral RNA and proteins, majority of the brain and spinal cord tissues exhibited minimal or no microscopic lesions. The virus tropism was restricted primarily to neurons, and virus particles (~61-68 nm) were present within axons of neurons and throughout the extracellular spaces. However, active virus replication was absent or minimal in majority of the brain and was limited to regions proximal to the olfactory tract. These data suggest that EEEV initially replicates in/near the olfactory bulb following aerosol challenge and is rapidly transported to distal regions of the brain by exploiting the neuronal axonal transport system to facilitate neuron-to-neuron spread. Once within the brain, the virus gains access to the ANS control centers likely leading to disruption and/or dysregulation of critical physiological parameters to produce severe disease. Moreover, the absence of microscopic lesions strongly suggests that the underlying mechanism of EEEV pathogenesis is due to neuronal dysfunction rather than neuronal death. This study is the first comprehensive investigation of EEEV clinical disease course and pathogenesis in a NHP model and will provide significant insights into the evaluation of countermeasure.Author SummaryEEEV is an arbovirus endemic in parts of North America and is able to produce fatal encephalitis in humans and domesticated animals. Despite multiple human outbreaks during the last 80 years, there are still no therapeutic or vaccines to treat or prevent human disease. One critical obstacle in the development of effective countermeasure is the lack of insights into EEEV pathogenesis in a susceptible animal host. We recently conducted a study in cynomolgus macaques to investigate the disease course by measuring clinical parameters relevant to humans. Following infection, these parameters were rapidly and profoundly altered leading to severe disease. In this study, we examined the potential mechanisms that underlie pathogenesis to cause severe disease. The virus was present in many parts of the brain and spinal cord, however, little or no pathological lesions as well as active virus replication were observed. Additionally, neurons were the predominant target of EEEV and virus transport was facilitated by axonal transport system to spread neuron-to-neuron throughout the brain and spinal cord. These data show that EEEV likely hijacks host cell transport system to rapidly spread in the brain and local/global neuronal dysfunction rather than death is the principal cause of severe disease.

BackgroundEbola 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.MethodsFourteen 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.ResultsDuration 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.ConclusionsWhile 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.

The West Africa Ebola virus disease outbreak of 2014-2016 demonstrated that responses to viral hemorrhagic fever epidemics must go beyond emergency stopgap measures and should incorporate high-quality medical care and clinical research. Optimal patient management is essential to improving outcomes, and it must be implemented regardless of geographical location or patient socioeconomic status. Coupling clinical research with improved care has a significant added benefit: Improved data quality and management can guide the development of more effective supportive care algorithms and can support regulatory approvals of investigational medical countermeasures (MCMs), which can alter the cycle of emergency response to reemerging pathogens. However, executing clinical research during outbreaks of high-consequence pathogens is complicated and comes with ethical and research regulatory challenges. Aggressive care and excellent quality control must be balanced by the requirements of an appropriate infection prevention and control posture for healthcare workers and by overcoming the resource limitations inherent in many outbreak settings. The Joint Mobile Emerging Disease Intervention Clinical Capability was established in 2015 to develop a high-quality clinical trial capability in Uganda to support rigorous evaluation of MCMs targeting high-consequence pathogens like Ebola virus. This capability assembles clinicians, laboratorians, clinical researchers, logisticians, and regulatory professionals trained in infection prevention and control and in good clinical and good clinical laboratory practices. The resulting team is prepared to provide high-quality medical care and clinical research during high-consequence outbreaks.

Ebola and Marburg are filoviruses and biosafety level 4 pathogens responsible for causing severe hemorrhagic fevers in humans with mortality rates up to 90%. The most recent outbreak in West Africa resulted in approximately 11,310 deaths in 28,616 reported cases. Currently there are no FDA-approved vaccines or therapeutics to treat infections of these deadly viruses. Recently we screened an FDA-approved drug library and identified numerous G protein-coupled receptor (GPCR) antagonists including antihistamines possessing anti-filovirus properties. Antihistamines are attractive targets for drug repurposing because of their low cost and ease of access due to wide use. In this report we identify common over the counter antihistamines, such as diphenhydramine (Benadryl) and chlorcyclizine (Ahist) as potential candidates for repurposing as anti-filovirus agents. Furthermore, we demonstrate that this potential is wide-spread through the 1(st) generation of H(1)-specific antihistamines but is not present in newer drugs or drugs targeting H(2), H(3) and H(4) receptors. We showed that the filovirus entry inhibition is not dependent on the classical antagonism of cell surface histamine or muscarinic acetylcholine receptors but occurs in the endosome, like the cathepsin inhibitor CA-074. Finally, using extensive docking studies we showed the potential for these drugs to bind directly to the EBOV-GP at the same site as toremifene. These findings suggest that the 1(st) generation antihistamines are excellent candidates for repurposing as anti-filovirus therapeutics and can be further optimized for removal of unwanted histamine or muscarinic receptor interactions without loss of anti-filovirus efficacy.

BACKGROUND: Several vaccine platforms have been successfully evaluated for prevention of Ebola virus (EBOV) disease (EVD) in nonhuman primates and humans. Despite remarkable efficacy by multiple vaccines, the immunological correlates of protection against EVD are incompletely understood. METHODS: We systematically evaluated the antibody response to various EBOV proteins in 79 nonhuman primates vaccinated with various EBOV vaccine platforms. We evaluated the serum immunoglobulin (Ig)G titers against EBOV glycoprotein (GP), the ability of the vaccine-induced antibodies to bind GP at acidic pH or to displace ZMapp, and virus neutralization titers. The correlation of these outcomes with survival from EVD was evaluated by appropriate statistical methods. RESULTS: Irrespective of the vaccine platform, protection from EVD strongly correlated with anti-GP IgG titers. The GP-directed antibody levels required for protection in animals vaccinated with virus-like particles (VLPs) lacking nucleoprotein (NP) was significantly higher than animals immunized with NP-containing VLPs or adenovirus-expressed GP, platforms that induce strong T-cell responses. Furthermore, protective immune responses correlated with anti-GP antibody binding strength at acidic pH, neutralization of GP-expressing pseudovirions, and the ability to displace ZMapp components from GP. CONCLUSIONS: These findings suggest key quantitative and qualitative attributes of antibody response to EVD vaccines as potential correlates of protection.

To evaluate potential immunocompetent small animal models of Zika virus (ZIKV) infection, we inoculated Syrian golden hamsters (subcutaneously or intraperitoneally) and strain 13 guinea pigs (intraperitoneally) with Senegalese ZIKV strain ArD 41525 or Philippines ZIKV strain CPC-0740. We did not detect viremia in hamsters inoculated subcutaneously with either virus strain, although some hamsters developed virus neutralizing antibodies. However, we detected statistically significant higher viremias (P = 0.0285) and a higher median neutralization titer (P = 0.0163) in hamsters inoculated intraperitoneally with strain ArD 41525 compared with strain CPC-0740. Furthermore, some hamsters inoculated with strain ArD 41525 displayed mild signs of disease. By contrast, strain 13 guinea pigs inoculated intraperitoneally with either strain did not have detectable viremias and less than half developed virus neutralizing antibodies. Our results support the use of the Syrian golden hamster intraperitoneal model to explore phenotypic variation between ZIKV strains.

Background Ebola virus disease (EVD) survivors are at risk for uveitis during convalescence. Vision loss has been observed following uveitis due to cataracts. Since Ebola virus (EBOV) may persist in the ocular fluid of EVD survivors for an unknown duration, there are questions about the safety and feasibility of vision restorative cataract surgery in EVD survivors. Methods We conducted a cross-sectional study of EVD survivors anticipating cataract surgery and patients with active uveitis to evaluate EBOV RNA persistence in ocular fluid, as well as vision outcomes post cataract surgery. Patients with aqueous humor that tested negative for EBOV RNA were eligible to proceed with manual small incision cataract surgery (MSICS). Findings We screened 137 EVD survivors from June 2016 – August 2017 for enrolment. We enrolled 50 EVD survivors; 46 with visually significant cataract, 1 with a subluxated lens, 2 with active uveitis and 1 with a blind painful eye due to uveitis. The median age was 24.0 years (IQR 17–35) and 35 patients (70%) were female. The median logMAR visual acuity (VA) was 3.0 (Snellen VA Hand motions; Interquartile Range, IQR: 1.2-3.0, Snellen VA 20/320 – Hand motions). All patients tested negative for EBOV RNA by RT-PCR in aqueous humor/vitreous fluid and conjunctiva at a median of 19 months (IQR 18-20) from EVD diagnosis in Phase 1 of ocular fluid sampling and 34 months (IQR 32-36) from EVD diagnosis in Phase 2 of ocular fluid sampling. Thirty-four patients underwent MSICS, with a preoperative median VA improvement from hand motions to 20/30 at three-month postoperative follow-up (P, Highlights • Ebola virus disease (EVD) survivors are at-risk for severe vision impairment due to uveitis and subsequent cataract development. • Fifty EVD survivors underwent ocular fluid sampling and tested negative for Ebola virus by RT-PCR. • Thirty-four survivors underwent cataract surgery with excellent safety measures and vision restorative outcomes. Ebola virus disease (EVD) survivors are at high risk for uveitis, an inflammatory condition affecting the eye that may lead to severe vision impairment, often from cataract. Because persistent Ebola virus (EBOV) has been identified within the immune privileged eye, understanding the prevalence of intraocular EBOV persistence is significant for patients, providers, and public health policy. In this study, 50 EVD survivors tested negative for EBOV by RT-PCR of their intraocular fluid. Thirty-four underwent cataract surgery with vision restorative outcomes. These findings improve our ability to impact vision care and quality-of-life for thousands of EVD survivors at-risk for eye disease.

During the 2013–2016 Ebola virus (EBOV) outbreak in West Africa, our team at USAMRIID evaluated the antiviral activity of a number of compounds, including favipiravir (T-705), in vitro and in mouse and nonhuman primate (NHP) models of Ebola virus disease. In this short communication, we present our findings for favipiravir in cell culture and in mice, while an accompanying paper presents the results of NHP studies. We confirmed previous reports that favipiravir has anti-EBOV activity in mice. Additionally, we found that the active form of favipiravir is generated in mice in tissues relevant for the pathogenesis of EBOV infection. Finally, we observed that protection can be achieved in mice down to 8 mg/kg/day, which is lower than the dosing regimens previously reported. An accompanying paper reports the results of treating nonhuman primates infected with EBOV or with Marburg virus with oral or intravenous favipiravir.

Although several COVID-19 vaccines have been developed so far, they will not be sufficient to meet the global demand. Development of a wider range of vaccines, with different mechanisms of action, could help control the spread of SARS-CoV-2 globally. We developed a protein subunit vaccine against COVID-19 using a dimeric form of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein as the antigen. We aimed to assess the safety and immunogenicity of this vaccine, ZF2001, and determine the appropriate dose and schedule for an efficacy study.We did two randomised, double-blind, placebo-controlled, phase 1 and phase 2 trials. Phase 1 was done at two university hospitals in Chongqing and Beijing, China, and phase 2 was done at the Hunan Provincial Center for Disease Control and Prevention in Xiangtan, China. Healthy adults aged 18-59 years, without a history of SARS-CoV or SARS-CoV-2 infection, an RT-PCR-positive test result for SARS-CoV-2, a history of contact with confirmed or suspected COVID-19 cases, and severe allergies to any component of the vaccine were eligible for enrolment. In phase 1, participants were randomly assigned (2:2:1) to receive three doses of the vaccine (25 μg or 50 μg) or placebo intramuscularly, 30 days apart. In phase 2, participants were randomly assigned (1:1:1:1:1:1) to receive the vaccine (25 μg or 50 μg) or placebo intramuscularly, 30 days apart, in either a two-dose schedule or a three-dose schedule. Investigators, participants, and the laboratory team were masked to group allocation. For phase 1, the primary outcome was safety, measured by the occurrence of adverse events and serious adverse events. For phase 2, the primary outcome was safety and immunogenicity (the seroconversion rate and the magnitude, in geometric mean titres [GMTs], of SARS-CoV-2-neutralising antibodies). Analyses were done on an intention-to-treat and per-protocol basis. These trials are registered with ClinicalTrials.gov (NCT04445194 and NCT04466085) and participant follow-up is ongoing.Between June 22 and July 3, 2020, 50 participants were enrolled into the phase 1 trial and randomly assigned to receive three doses of placebo (n=10), the 25 μg vaccine (n=20), or the 50 μg vaccine (n=20). The mean age of participants was 32·6 (SD 9·4) years. Between July 12 and July 17, 2020, 900 participants were enrolled into the phase 2 trial and randomly assigned to receive two doses of placebo (n=150), 25 μg vaccine (n=150), or 50 μg vaccine (n=150), or three doses of placebo (n=150), 25 μg vaccine (n=150), or 50 μg vaccine (n=150). The mean age of participants was 43·5 (SD 9·2) years. In both phase 1 and phase 2, adverse events reported within 30 days after vaccination were mild or moderate (grade 1 or 2) in most cases (phase 1: six [60%] of ten participants in the placebo group, 14 [70%] of 20 in the 25 μg group, and 18 [90%] of 20 in the 50 μg group; phase 2: 37 [25%] of 150 in the two-dose placebo group, 43 [29%] of 150 in the two-dose 25 μg group, 50 [33%] of 150 in the two-dose 50 μg group, 47 [31%] of 150 in the three-dose placebo group, 72 [48%] of 150 in the three-dose 25 μg group, and 65 [43%] of 150 in the three-dose 50 μg group). In phase 1, two (10%) grade 3 or worse adverse events were reported in the 50 μg group. In phase 2, grade 3 or worse adverse events were reported by 18 participants (four [3%] in the two-dose 25 μg vaccine group, two [1%] in the two-dose 50 μg vaccine group, two [1%] in the three-dose placebo group, four [3%] in the three-dose 25 μg vaccine group, and six [4%] in the three-dose 50 μg vaccine group), and 11 were considered vaccine related (two [1%] in the two-dose 25 μg vaccine group, one [1%] in the two-dose 50 μg vaccine group, one [1%] in the three-dose placebo group, two [1%] in the three-dose 25 μg vaccine group, and five [3%] in the three-dose 50 μg vaccine group); seven participants reported serious adverse events (one [1%] in the two-dose 25 μg vaccine group, one [1%] in the two-dose 50 μg vaccine group, two [1%] in the three-dose placebo group, one [1%] in the three-dose 25 μg vaccine group, and two [1%] in the three-dose 50 μg vaccine group), but none was considered vaccine related. In phase 2, on the two-dose schedule, seroconversion rates of neutralising antibodies 14 days after the second dose were 76% (114 of 150 participants) in the 25 μg group and 72% (108 of 150) in the 50 μg group; on the three-dose schedule, seroconversion rates of neutralising antibodies 14 days after the third dose were 97% (143 of 148 participants) in the 25 μg group and 93% (138 of 148) in the 50 μg group. In the two-dose groups in phase 2, the SARS-CoV-2-neutralising GMTs 14 days after the second dose were 17·7 (95% CI 13·6-23·1) in the 25 μg group and 14·1 (10·8-18·3) in the 50 μg group. In the three-dose groups in phase 2, the SARS-CoV-2-neutralising GMTs 14 days after the third dose were 102·5 (95% CI 81·8-128·5) in the 25 μg group and 69·1 (53·0-90·0) in the 50 μg group.The protein subunit vaccine ZF2001 appears to be well tolerated and immunogenic. The safety and immunogenicity data from the phase 1 and 2 trials support the use of the 25 μg dose in a three-dose schedule in an ongoing phase 3 trial for large-scale evaluation of ZF2001's safety and efficacy.National Program on Key Research Project of China, National Science and Technology Major Projects of Drug Discovery, Strategic Priority Research Program of the Chinese Academy of Sciences, and Anhui Zhifei Longcom Biopharmaceutical.For the Chinese translation of the abstract see Supplementary Materials section.

Background Residents of long-term care facilities (LTCFs) have been prioritised for COVID-19 vaccination because of the high COVID-19 mortality in this population. Several countries have implemented an extended interval of up to 12 weeks between the first and second vaccine doses to increase population coverage of single-dose vaccination. We aimed to assess the magnitude and quality of adaptive immune responses following a single dose of COVID-19 vaccine in LTCF residents and staff. Methods From the LTCFs participating in the ongoing VIVALDI study (ISRCTN14447421), staff and residents who had received a first dose of COVID-19 vaccine (BNT162b2 [tozinameran] or ChAdOx1 nCoV-19), had pre-vaccination and post-vaccination blood samples (collected between Dec 11, 2020, and Feb 16, 2021), and could be linked to a pseudoidentifier in the COVID-19 Data Store were included in our cohort. Past infection with SARS-CoV-2 was defined on the basis of nucleocapsid-specific IgG antibodies being detected through a semiquantitative immunoassay, and participants who tested positive on this assay after but not before vaccination were excluded from the study. Processed blood samples were assessed for spike-specific immune responses, including spike-specific IgG antibody titres, T-cell responses to spike protein peptide mixes, and inhibition of ACE2 binding by spike protein from four variants of SARS-CoV-2 (the original strain as well as the B.1.1.7, B.1.351, and P.1 variants). Responses before and after vaccination were compared on the basis of age, previous infection status, role (staff or resident), and time since vaccination. Findings Our cohort comprised 124 participants from 14 LTCFs: 89 (72%) staff (median age 48 years [IQR 35·5–56]) and 35 (28%) residents (87 years [77–90]). Blood samples were collected a median 40 days (IQR 25–47; range 6–52) after vaccination. 30 (24%) participants (18 [20%] staff and 12 [34%] residents) had serological evidence of previous SARS-CoV-2 infection. All participants with previous infection had high antibody titres following vaccination that were independent of age (rs=0·076, p=0·70). In participants without evidence of previous infection, titres were negatively correlated with age (rs=–0·434, p

Zika virus (ZIKV) is a mosquito-borne member of the Spondweni complex of the genus Flavivirus, family Flaviviridae. The genus Flavivirus comprises many important human pathogens such as Saint Louis encephalitis, yellow fever virus, dengue virus (DENV1–DENV4), West Nile virus, Japanese encephalitis virus, and tick-borne encephalitis virus.1 The ZIKV genome comprises a single-strand, positive-sense RNA of approximately 11 kb in length. The genome consists of a 5′ untranslated region (UTR) (∼107 nt), an open reading frame (ORF) ∼10.2 kb in length, and a 3′ UTR (∼428 nt). The single ORF encodes three structural proteins: capsid (C), premembrane/membrane (prM), envelope (E), and seven non–structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5).1 Similar to other flaviviruses, ZIKV enters cells through receptor-mediated endocytosis, and the low endosomal pH induces fusion to release the genomic RNA into the cytoplasm. The genomic RNA is translated into a single polyprotein that is processed to facilitate replication, virion assembly, and release of mature virions. The mature ZIKV virion structure is similar to that of other flaviviruses, comprising a nucleocapsid core consisting of C protein and genomic RNA and an icosahedral shell consisting of 180 copies of E and M proteins (or prM) embedded in a host-derived lipid bilayer.2 ZIKV was first isolated from the blood of a sentinel rhesus macaque in 1947 in the Zika Forest in Uganda.3 In the following year, ZIKV was isolated from pools of Aedes africanus mosquitoes, and subsequent studies demonstrated virus transmission by Aedes aegypti to both mice and rhesus macaques.4 In the following decades (1950s–1980s), ZIKV was isolated from humans and multiple mosquito species in sub-Saharan Africa and Southeast Asia, and suggesting maintenance in both sylvatic (primarily Aedes spp. mosquitoes and nonhuman primates [NHPs]) and urban cycles (mainly A. aegypti and humans).5–19 ZIKV consists of a single serotype, with isolates comprising two geographic lineages (African and Asian) that have caused sporadic or underreported human outbreaks.20,21 However, starting in 2007, outbreaks were reported in the Yap island in the Federated States of Micronesia and in Gabon, and by 2013 ZIKV was imported from French Polynesia into northeast Brazil resulting in the largest reported epidemic to date and its subsequent expansion to many parts of Americas including mainland USA (Florida and Texas).22–27 Since the start of the 2007 epidemic, ZIKV has spread to or been detected in 84 countries and territories in the Americas, Asia, and Africa, with reported evidence of autochthonous transmission (http://apps.who.int/iris/bitstream/10665/254714/1/zikasitrep10Mar17-eng.pdf?ua=1). Most human ZIKV infections are asymptomatic.28,29 Symptomatic infections typically present as a self-limiting acute febrile illness with symptoms ranging from fever, headache, myalgia, and rash; however, severe clinical manifestations including congenital microcephaly and other fetal defects, Guillain–Barre syndrome (GBS), and death have been reported.30,31 Although ZIKV is transmitted primarily through mosquito/human cycle, the virus can also be transmitted through sexual contact and blood transfusions.32–34 Before its arrival in the Americas, ZIKV was an obscure and understudied pathogen; as a consequence, few virus isolates were accessible for research and vaccine/therapeutic development. In addition, little or no basic in vitro and/or in vivo characterization data were available on accessible isolates. In the present study, we investigated the potential phenotypic differences between low-passage African (ArD 41525) and Asian isolates (CPC-0740 and SV0127-14) in vitro and in vivo.

INTRODUCTION During the 2014-2016 Ebolavirus (EBOV) outbreak, several candidate therapeutics were used in EBOV-infected patients in clinical trials and under expanded access for emergency use. This review will focus briefly on medications used during the outbreak. We will discuss current therapeutic candidates and their status and will then turn to a related and essential topic: supportive care and the standard of care for filovirus infected patients. Potential benefits and pitfalls of combination therapies for filoviruses will be discussed. Areas covered: Clinical trials of therapeutics targeting EBOV; clinical usage of therapeutics during recent EBOV outbreak; potential need for combination therapy; role of supportive care in treatment of Ebola virus disease (EVD). Expert commentary: In the absence of another large scale EBOV outbreak, the path to therapeutic product licensure in the United States of America (USA) would need to be via the FDA Animal Rule. However, human data may be needed to supplement animal data. The future of filovirus therapeutics may therefore benefit by establishing the ability to implement clinical trials in an outbreak setting in a timely fashion. Supportive care guidelines for filovirus infection should be defined and established as standard of care for treatment of EVD.

Filoviruses, consisting of Ebola virus, Marburg virus and Cuevavirus, cause severe hemorrhagic fevers in humans with high mortality rates up to 90%. Currently, there is no approved vaccine or therapy available for the prevention and treatment of filovirus infection in humans. The recent 2013-2015 West African Ebola epidemic underscores the urgency to develop antiviral therapeutics against these infectious diseases. Our previous study showed that GPCR antagonists, particularly histamine receptor antagonists (antihistamines) inhibit Ebola and Marburg virus entry. In this study, we screened a library of 1220 small molecules with predicted antihistamine activity, identified multiple compounds with potent inhibitory activity against entry of both Ebola and Marburg viruses in human cancer cell lines, and confirmed their anti-Ebola activity in human primary cells. These small molecules target a late-stage of Ebola virus entry. Further structure-activity relationship studies around one compound (cp19) reveal the importance of the coumarin fused ring structure, especially the hydrophobic substituents at positions 3 and/or 4, for its antiviral activity, and this identified scaffold represents a favorable starting point for the rapid development of anti-filovirus therapeutic agents.

Opinion statement Purpose of review Ebola virus, a member of the Filoviridae family, is a causative agent of severe viral hemorrhagic fever in humans. Over the past 40 years, the virus has been linked to several high mortality outbreaks in Africa with the recent West African outbreak resulting in over 11,000 deaths. This review provides a summary of the status of the drug discovery and development process for therapeutics for Ebola virus disease, with a focus on the strategies being used and the challenges facing each stage of the process. Recent findings Despite the wealth of in vitro efficacy data, preclinical data in animal models, and human clinical data, no therapeutics have been approved for the treatment of Ebola virus disease. However, several promising candidates, such as ZMapp and GS-5734, have advanced into ongoing clinical trials. Summary The gravity of the 2014-2016 outbreak spurred a heightened effort to identify and develop new treatments for Ebola virus disease, including small molecules, immunotherapeutics, host factors, and clinical disease management options. Disclaimer Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endoresed by the U.S. Army.

Recent Ebola virus (EBOV) outbreaks in West Africa and the Democratic Republic of the Congo have highlighted the urgent need for approval of medical countermeasures for treatment and prevention of EBOV disease (EVD). Until recently, when successes were achieved in characterizing the efficacy of multiple experimental EVD therapeutics in humans, the only feasible way to obtain data regarding potential clinical benefits of candidate therapeutics was by conducting well-controlled animal studies. Nonclinical studies are likely to continue to be important tools for screening and development of new candidates with improved pharmacological properties. Here, we describe a natural history study to characterize the time course and order of progression of the disease manifestations of EVD in rhesus monkeys. In 12 rhesus monkeys exposed by the intramuscular route to 1000 plaque-forming units of EBOV, multiple endpoints were monitored for 28 days following exposure. The disease progressed rapidly with mortality events occurring 7&ndash, 10 days after exposure. Key disease manifestations observed consistently across the infected animals included, but were not limited to, viremia, fever, systemic inflammation, coagulopathy, lymphocytolysis, renal tubular necrosis with mineralization, and hepatocellular degeneration and necrosis.

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., Author summary Zika virus (ZIKV) is primarily maintained in an enzootic cycle involving nonhuman primates and mosquitoes, with epizootics and epidemics occurring when the virus is introduced into naïve populations of nonhuman primates or humans, respectively. While, the primary transmission mechanism of the virus is by the bite on an infected mosquito, ZIKV can also be sexually transmitted. In an effort to develop novel animal models to study ZIKV disease, and to better understand the role of nonhuman primates as amplification and maintenance hosts of ZIKV in nature, we modeled mosquito-borne and sexual transmission of ZIKV in the enzootic host, the African green monkey (AGM). Infection dynamics and neutralizing antibody responses in all three AGM ZIKV models (subcutaneous, intravaginal and intrarectal) in the absence of clinical illness–recapitulated reported generalized human disease course. Furthermore, we detected prolonged shedding with high viral loads and infectious virus in the vaginal swabs collected from an infected female AGM inoculated intrarectally. Notably, these results support limited human clinical evidence that ZIKV transmission can occur during female-to-male vaginal sexual acts, and furthermore indicate the existence of ZIKV super-spreaders. Finally, our results indicate sexual transmission of ZIKV could occur among infected nonhuman primates (e.g. Chlorocebus spp.) in Africa and may serve as a secondary transmission and maintenance mechanism in the absence of mosquito-to-nonhuman primate transmission.